(SANITIZED)UNCLASSIFIED SCIENTIFIC PAPERS ON IMMUNE REACTIONS INVOLVING ANTIGENIC STIMULUS(SANITIZED)

• Document Type: CREST
• Collection: General CIA Records
• Document Number (FOIA) /ESDN (CREST): CIA-RDP80-00247A004200090001-4
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• Original Classification: U
• Document Page Count: 291
• Document Creation Date: December 23, 2016
• Document Release Date: January 14, 2014
• Sequence Number: 1
• Publication Date: August 12, 1964
• Content Type: REPORT

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Developtent of Immune Reactions in the Absence or Presence
of an Antigenic Stimulus
aerzl J. Mandel L., Miler I., Lha I.
Department of Immunology, Institute of Microbiology,
Czechoslovak Academy of Sciences, Prague, Czechoslovakia
The aim of this work was to di.irferentiate serum factors
with some immune activites, which develop spontaneously
without dependence on an antigenic stimulus,, from antibodies
which arise in response to the injection of antigen. Under
normal conditions(conventionally reared animals) antigenic
stimuli are practically uncontrollable; they act in the
or:;anism, such as bacteria colonizin,]; the intestine, respi-
ratory tract and surface of the body. Moreover, food contains
antigenic substances which are also a source of antigenic
. stimuli. Antibodies arising in response to them, without
interference anl control, are called natural antibodies.
On the other hand, natural factors are present in the serum
which have the capacity of interacting with macromolecular
substances (e.g. alpha macroglobulin reacting with insulin,
natural conglutiain with zymosan (1')). In this communication,
the role of antigen in developing of the serum factors above
mentioned is sttidied.
Experimental model. The newborn animals contain anti-
bodies transferred from the mother and a great number of
_antigenic substances stimulating the maturing of its own
immune mechanisms. The level of transferred antibodies does
not usually permit detection of antibodies formed by the
young animal itself. It is, therefore, of advantage to use
a type of animal whose placental barrier does not permit the
transfer of antibodies into the circulation of the foetus.
If such animals (e.g. piglets) are artificially fed and do
not obtain maternal colostrum, we do not find antibodies
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passively transferred from the mother in their serum.
In order further to exclude contact of the newborn animal
with microbial antigens, the foetus is removed from the
mother before birth under sterile conditions an put into
an incubator where it is excluded from bacterial contami-
nation.(Figs 1, 2, 3). However, the rearing of animals
without microorganisms does not fully guarantee that anti-
genic stimuli are completely eliminated, because of cont@nt-
063 dead bacteria and other antigenic components id food.
We have attempted to reduce this source of antigenic stimuli
by using a nonantigenic diet (mixture of amino acids and
vitamins) and also by observing the development of immune
factors in the early postnatal period, when immune response
? to small doses of antigen is reduced. We consider that
, studies of natural and immune factors in animals which
have not obtained antibodies by transfer from the mother
anl are reared under sterile conditions in th3 early stages
of ontocenesis,is the best model for solving these questions
-at the present time.
The characterization Of some serum fractions of sterile
precolostral piglets. The electrophoretic pattern 'of pig-
let sera shows, in agreement with published results, that
the newborn do not possess serum ..-globulin. After concen-
tration, however, protein has been d:2tected in the region
corresponding to ',7globulin (2). Sera of precolostral pig-.
? lets contain approximately 40 pg per ml. of this protein.
T-ie incorporation of methionine into the :;?;-globulin fract-
ion of the newborn was demonstrated by the use of labeled
methionine S35. The rate of increase in radioactivdty
indicated the typical process of synthesis .(3). The nature
of_l':-globuldn in the :newborn was studied by physico-chemi-
cal and immunochemical methbds. Two fractions were separa-
, ted on DEAE cellulose,. A fraction .(I) of the neonatal
r-globulin is not precipitated'and has a sedimentation
coefficient of 2.7 S. The other fraction (II) is precipitated
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with.antiserum against the, 1.--globulin of adult pigs and
has a sedimentation coefficient of 5.1 S (4). This
fraction bears no relation' to Bence Jones protein but
a relation between 5 S neonatal 9-globulin and H chains
of adult pig r-globulin was determined by the fingerprint
technique (5).
The evidence that antibodies are not present irl_pEt:
colostrallet. Antibodies have not hitherto been
demonstrated by any serological reaction to any bacterial,
virus, phage or tissue antigens. No antibodies were detect-
ed by the agglutination reaction to the 0 and H antigens
of various Gram-negative bacteria, nor by passive haemag-
glutination' with antigens isolated from bacterial strains
and bound on. to sheep erythrocytes (6).
Passive haemolysis was carried out with erythrocytes,
to which different antigens were adsorbed, not only with
usual erythrocyte concentration (1%) but with progressively
decreasing concentrations of erythrocytes (0.001%) (Fig.4).
In this way the amount of antibodies necessary to produce
haemolysis was greatly decreased, to the level l06ig N/ml
(7)e The bactericidal reaction for which strains in the
S-forir, were selected, has the same degree of sensitivity
(8). Even detection of antibodies by rTspnizatior of strains
in the S-form (at the level of 10-6 pg. N/ml) with sera of
newborn precolostral piglets was negative. The sera do not
display neutralization activity to phage (the experiments
made by Dr.Trnka) neither neutralization activity
against poliomyelitis viruses (Dr.Slonim). Antibodies were
not only not found in the serum but also not in the
concentrated v-globulin fraction even on using passive
haemagglutination and passive haemolysis with adsorbed
diphtheria toxoid as in the experiments of Segre (9).
We conclude, therefore, that the protein of the .-globulin
type in precolostral newborn piglets has not any detectable
antibody activity.
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Although antibodies were not aetected in newborn
piglet sera, a certain amount of complement was demon-
strated immediately after birth by the haemolytic reaction
(about 2 C'H50 units). The amount of complement increased'
gradually ih animals reared under sterile conditions;
20-day old piglet had 15 - 20 C'H units/ml. (9).
The immunological properties of piglet sera without
the presence of' antibodies. Some chanaes in antigen
suspension similar to antigen-antibody reaction can be
observed without the presence of antibody: bacterial agglu-
tination at low pH, haemolysis by complement of erythrocytes
previously treated with tannic acid, etc. The data will be
presented that complement can act on certain bacterial
surfaces even in the absence of antibodies.
a. Baetericidal reaction: In experiments using strains
in the typical S-form, e.g. strain of Salmonella paratyphi B,
we never detected bactericidal activity in precolostral
piglet sera. On testing a larger group of Gram-negative
bacteria, however, we found that thevasera exert, a-bacte-
ricidal effect on some bacterial strains. These strains,
which were sensitive to piglet complement, were in the
typical R-form: On absorbing sera at 00 with R strains
the bactericidal effect of serum waa not abolished; it was
abolished, however, if any components of complement was?
inactivated. In selected strains it was found that there is
a direct relationship between the character of their sur-
faces (R-form) and the degree of dilution of test serum
still capable of bactericidal activity (Table 1). We-further
demonstrated that there is a dependence of the amount of
piglet complement, the character of the bacterial surface
and the bactericidal effect: sera with a small amount of
complement only have an effect on strains in the R-form
(Table a). Higher-complement levels (5 C'H50 units) act
against strain 346 which has the characteristics of bacteria
in the Solform- and which is not sensitive to sera with
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a lower complement level. Bactericidal activity is thus,
determined by the surface of the bacteria and not by its
specific antigenic structure (the series included strains
of coli, S.typhi, Shigella shigae, etc.). At the same time
as using precolostral piglet serum we also worked with
precolostral calf sera. It was shown that their bacterici?
dal effect against some strains, e.g. Shigella shigae,
is higher than would have been expected from the relation-
ship between calf and piglet serum (Fig. 5). We therefore
used different serological metheds to determine whether
antibodies to the Shigella strain were not present in the
calf serum. We found that with 0 antigen adsorbed to
erythrodytes (antibodies to this antigen are responsible
for the bactericidal reaction) and passive haemolysis
done with minimal numbers of erythrocytes (0,001%) the '
demonstration of antibodies by this method is more sensitive
than the bactericidal reaction (T.3). We conclude that if
antibodies are demonstrated in newborn serum by the bacte-
ricidal reaction only and not by the more sensitive passive
haemolysis with minimal amounts of erythrocytes, precolo-
stral serum probably does not contain antibodies. We further
found that the bactericidal activity of precolostral serum
can only be abolished if it is absorbed with zymosan in
the presence of complement. If native calf serum is treatel
with zymosan, the bactericidal action disappears, after
adding piglet complement to the absorbed calf serum in a
concentration which does not itself have a bactericidal
effect, bactericidal activity was not changed and the
bactericidal effect was not present. On the contrary, in
calf serum first inactivated at 56oC and treated with (Fig.6)
.zymosan to which the same amount of piglet complement had
been added after inactivation, bactericidal activity was
almost fully restored. Our contemporary programme is to
establish vhether absorption of sera does not affect some
components of complement which were restored on adding
neonatal complement or whether precolostral sera contain
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a substance with properties such as properdin or natural
conglutinin.
?
b. Opsonizing effect of precolostral piglet serum:
A study was made of the opsOnizing activity of neonatal
precolostral piglet serum to strains in the S- and R-form.
-Phagocytosis was determined by perfusion of the isolated
liver i0) and by the method of determining the time course
of bacterial clearance from the circulation in neonatal
piglets. Strains, which are in the typical S- and R-form
weresselected for the study of opsonization (Table 1).
The concentration of the bacterial suspension for opsoni-
zation was 5 x 104 bacteria/ml. 0.1 ml of this suspension
was mixed with 0.9 ml. test serum and left for one hour
in the refrigerator. Bacteria were introduced into the
'afferent cannila leading to vena portae and the number of
up
bacteria not taken by the liver was determined in
samples taken_off from tha inferior vena cava. Opsonization
was carried out in the same way in clearance tests in
vivo and 105 bacteria/kg. body weight were injected into
the blood stream of piglets. The bacterial count was deter-
mined in blood samples obtained by cardiac puncture at
different time intervals after injection.
In the first experiments on the isolated rat liver
it was found that the uptake of E.coli suspended in Ringer
splUtion is.dependent on the character of the bacterial
surfaces. An average of only 10% of the S-form are retained,
whereas in the R-form the number retained amounts to 55'- 64%.
In experiments in which E.coli (S-form) were opsonized with
neonatal serum containing 2 - 3 units of C H50 complement,
it was found that complement has an opsonizing effect and
increases the number of bacteria retained from 10 to 48 %.
Any inactivation of complement components abolished the
opsonizin activity (Table 4) (11).
In further experiments phagocytosis was determined
by the method of clearance of bacteria from the blood stream
in neonatal precolostral piglets. Evidence was provided that
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E.6,oli in the. S-form is riot phagocYtosd in the heWborn
(Fig:7)i"0n the other harid if the R-form of'the'strain
-is.A.njected into neOnatal piglets it is effectively ?
cleared from the bleod stream (Fig.8.).. In order to deter-
mine the minimal amount of antibodies for opsonization and
phagocytosis of 064 in the S-form, we mixed a bacterial
suapehsiOn in the S-form_with different amounts of rabbit.
antibody. It was found that dilutions of 1078 - 1079
eP,.antiseruM'still.produde a full opsonizing effect die- .
played by the:complete clearance of the ihjected bacteria
from the blood stream. Dilutions of 10710 and 10711 lead
to only a partial and transient decrease of the number
-of circulating bacteria (Fig.9). /Since the same strain
miXed with neonatal Serum containing a small amount of
? complement did not have an opsonizing effect it shows
that the precolostral sera of neonatal piglets does not
:-contain antibodies detectable by this very sensitive .
test (by which antibodies. are determined in a concentra-
tion of abOut,1076g N/m1). This experiment, therefore,
- shows that the sera of animals in which no antibodies
have been demonstrated by any method, contain complement
/which acts on some bacterial surfaces and is thus able
.to imitate immune processes for which the presence of
..antibodies has hitherto been considered to be necessary., ?
The developpent.of.antibodv formation in sterile
animals after immunization with different antigens:.
. The study of antibody' formation in sterile.precolostral
piglets has a number of advantages. Since there is no
-.transfer of rglobulin from the mother. and there is no
level of natural and passively acquired antibodies, the
first antibodies detected are formed by. the'infant animal
itself..Since.the sarum contains only a, small amount Of -
'neonatal. qglobulin of low molecular weight, the character
of the first antibodies -can te well determined..
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a.- Time course of onset of antibody formation after
imMunization with .different antigens:.Newborn precolostral
piglets were given an injection of Soparatyphi B and anti-
bodies detected mainly using the' bactericidal reaction
? ' --
permitting detection of 196 eg of. antibody N/ml. The new-
born animals were siven'i.p. 'injections of the maximum
'dose 'of antigen that .they tolerate, i.e. 2.5 x 1010 bacteria
:in 5 ml. The first antibadiea were but rarely detected
5:days after immunization.. The amount, of antibody increased
to the 10 - 15th. day after the injection of antigen. If
sheep erythrbcytes are injected (20% suspension in amounts
of 10 or 20 ml) antibodies appear earlier, in most animals
on the fifth day after the injection of antigen. However,
? .
we did not succeed in demonstrating antibody on the third'
-;(day after injection, even- using very sensitive tests. For
demonstrating antibodies by the haemagglutination reaction
we use a 0.1% suspension of erythrocytes, for demonstrating
antibodies by the haemolytic .reaction a 0.001% suspension.
,Very.similar results - i.e. ?negative demonstratiaa.of anti-
.bodies on the third dayaft"er the injection .of antigen
- and reliable demonstration on the fifth day - were obtained
. after immunization with the corpuscular antigen of phage T2
and. virus (Sabin attenuated vaccine strain). A comParison
of the results after immunization \Ath the different anti-
gens-is given in:the table 5, Fig.' 10. It is evident that'
the smallest immunizing effect was displayed by the bacte7
. rial antigen _Separatyphi B. If it cannot be objected that
the different.results.in the formation of antibodies express
.different sensitivity of the serological reactions'to all
antigens (we used tests whose sensitivity-was at the level
of 10--6 kigN/m1) thenit'is necessary to consider the quality
and quantity of injected antigen.
? It is very difficult to compare the amount of antigenic
-substance injected in the complex structure Of corpuscular
.antigens since haemolytic, -haemagglutinating and virus and
phage neutralizing antibodies only react with a certain
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part of the whole injected corpuscle. WeiltherefOre,
wished to know whether the previously observed finding
'thatl increasing the amount of antigen speeded up the onset
of antibody formation in young animals (12) would be valid
for neonatal piglets. From the results (Fig.11) it is
evident that inereasing the amount of injected erythrocytes
and phage (Fig.10) led to the earlier onset of antibolly
formation and also to a higher resulting titre. .
The effect of the passive transfer of antibodies for
the Onset of antibody. formation in infant animals has often
been discussed and investigated in a number of works. Most
work has shown that the transfer of antibodies delays the
onset of immUnization processes. It was found only rarely
that the passive transfer of antibodies has a stimulating
? effect. In the work of Segre (13) on the same model of
precolostral piglets, the passive transfer of antibodies,
was considered to be the basis of a good onset of an immunity
response in infant animals. In all the antigens used we
. obtained a good response in precolostral piglets. de made
a comparison of the immunizing effect of the same amount
of erythrodyte-antigen in piglets reared conventionally
with the mother and in sterile precolostral piglets. The
. table shows that the passive transfer of antibodies inhibits
t to some extent the onset of the actual immunizing process
in infant animals (Table 5, Fig. 12).
de further wished to find out the e,..2rliest onset of
antibody formation and whether there is an increased immune
response to different antigenic stimuli during the maturing
of the individual in infant animals reared non exposed to
antigens. We were unable tO detect antibody formation after
immunizing the foetus in utero one month before term
(gestation lasts three months in pigs) using Soparatyphi B
::and Brucella suis as antigens; these results were published
in 1960 (6). In recent years we have used sheep ef.ythro-
cytes for intrauterine immunization and have demonstrated
antibody formation immediately after birth in piglets
, immunized one month before term. In some animals, however,
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antibodies Were -not :detected at birth after intrauterine
: immunization.. This codid have ?be.en because the level of
?
antibodies formed was not high enough or ?because
antibo-
:'di?:which could, have ..been detected during the Month dis-
appeared before birth. -If ?Puch an animal is giVen the same
? injection of- antigen immediately after birth as is given
..ito newborn animals not immunized in utero,. the onset, course
?i and level Of antibedy 'formation has the typical character
:of a secondary reaction' ( table 7). If the same dose of
antigen as the first immuni.ins dose (10 ml. of 20% sheep
erythrocytes) is injected into young animals of _various. ? ,
;ages- reared under Sterile conditions, we do not observe a
significant increase in the, immunizing 'effect which would
? show 'that ?marked. changes in the ability to respond to anti-
gen luring ontogenesis occur without an antigenic stimulus.
.:?This 'finding has obviously a parallel in the number of
:.antibody pro lucin,3 cells detected-, as will be reported
ib. Characterization of the first antibodies formed:
: A determination.' was made of the molecular weight of the
? 'first antibodies in the sera of precolostral -sterile piglets.
Antibodies to erythrocytes,: phage and strain S.paratyphi B
were _centrifuged in a5haroegradient in ,a Spinco rotor
:40 centrifuge 'at 35,000 rop.m. for. 16 hours. Six layers
' were .successively separated in which the bactericidal,
? haemolytic and neutralizing activity of antibodies were
? tested. In all samples, antibodies were found in the last,-
, i.e. the fifth and:sixth fractions at the bottorri of the tube.
These. tests confirmed _a whole series of. similar work that
.? the first formed antibodies in newborn animals are of the
macromolecular type (14,3?5)..,
.The first, detectable antibodies howeVer, are not
characterized ,by this property only, but also by serological
? properties. If antibodies to erythrocytes are determined by
? the test described, in which the sensitivity is increased
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lowering:the :erythrocyte concentration, we do not
find that the increased sensitivity of the aerological
reaction. has a:linear dependence as in the sera of hyper-
'immune adultanimala (Fig.13). Tne.results,show that.
infant antibodies have a,different binding capacity from
that of the antibodies ef.hyperimmune adult animals. This
:could be due to the fact that they are Mainly 19 S anti-
. .
bodies. We therefore isolated antibodies of the 19 S
,character from adult hyperimmunized animals on Sephadex
? G7200 (16) and determined the dependence of the amount
of antibody essential for 50% haemolysis at different
concentrations of erythrocytes. In 19 S antibodies of
? adult-animals we found a linear dependence, i.e. within
the class of one molecular type of antibody the binding
- capacity changes. This was also confirmed for 7 S anti-
bodies. The antibodies formed immediately after birth .
during the secondary reaction, according to their sedimen-
tation characteristics in a.sacharose gradient, are anti-
bodies of the 7 S type. These antibodies however, when
tested for binding, capacity by the test using a decreased
number of erythrocytes, also failed to show a linear
dependence. Again the reverse to 7 S antibodies of adult
,animais.
Differences in the binding capacity in the first
antibodies of immunized pigleta. were found with anti-phage
sera. It was found that the antibodies have a very small
neutralizing capacity,. which, hewaver, is considerably(/,)
increased if complement is added to the.test. The source
of complement Was the serum from precolostral non immunized
piglets which ,itself has no neutralizing activity to phage.
Here complement-evidently acts as a cofactor which strength-
ens the binding of easily dissobiable antibodes on the
phage particle similarly as in the first antiviral antibodies
described' by.Dulbecco (17).
,
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A further characteristic property of the first anti-
bodies in newborns is their:..different.sensitivity :to
.thercai)toethano1 (ME). Hyperimmune sera were tested and
it was found that different concentrations of ME used
by so Me authors (18; .19), for the inactivation,of 19 S
antibodies (11,M, 0.1 M, 0,05 M). do not exert a different
effect. After the action of these- concetrationa we found.
the same titres, i.e. the same decrease in the initial
haemagglutination reaction.: If, however, we inveStigate
the first antibodies of infant animals,: the serological ?
activity of antibodies disappeared after treatment with
the given concentrations of mercaptoethanol. If sera obtain-
ed from infant animals reacting after birth with -a-secon-
dary reaction (i.e. those which were immtanized in utero)
,are treated with 0.05.M ME, the titre of antibodies is
only partly decreased. However, 1 M concentration of ME,
which has the same effect on hyperimmune sera as 0.05 M ME
completely abolishes the serological activity of infant
7 S antibodies.
.The results obtained with immunization of the foetus
and newborn with different antigens provide evidence that
. the Character ,of the antigen is of importance for the
?; stimulation of the antibody response as well as the amount
of. antigen injected. Unlike the proteins found in the
? serum of nonimmunized newborn piglets in the rglobulin
zone, wEich have a low molecularweight of about 5 SI the
first antibodies formed are macromolecular antibodies
with different binding capacities from those of adult
r
ahimals. Newborn piglets are able to react with a secondary
reaction immediately after birth if the first dose is
given in utero. These antibodies appear to be of the .7 S type.
1-But their properties still show some characteristics of
primitive antibodies (low binding capacity, sensitivity
.to mercaptoethanol). It can therefore be assumed that the
change in molecular type, like increased resistance to ME
. and changes in binding capacity, will not occur at once ?
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-but that there will evidently be further antibodies of
intermediate mOleCular Characteristics.
The present results permit a more exact distinction
between the antibody and nonantibody natural components
-of sera. Antibody is not only substance bound to a certain
group of proteins of. sera (according to present knowledge
to r-globulin) but is the result of adaptation processes
the'organist after contact with antigen. The adaptation
process is characterized by the formation of molecules
of increasing fitness and effectiveness in their reaction
with antigen. It is just these properties that .distinguish
'antibody from thenCnantibody components of the serum which
are present and have a certain binding capacity with some
macromolecular substances - even imitating the reaction of
antigen with antibody - but which are formed spontaneously
and their properties do not undergo any change after
antigen injection.
? .
?
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Lachman,P.J.-: Immunoogy 687,(1962)
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5.Fran?F. and tiha,I.:Immunochemistry 1_: 49 (1964).
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7.,&terz1,J. and Kostka J.: Folia microbio1.8 : 60 (1963)
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9...Meyers,W.M. and Segre,D.: J.Immunol. 91 ?: 697 (1963)
10..Howard,J.G., Wardlaw,A.C.: Immunology 1,: 338 (1958)
11.'8terzl J. Folia micrObiol. 8 : 240 (.1963)
12...terz1:, J. and Trnka, Z.; Nature 179 : 918 (1957)
13. .Segre,D. and Kaeberle,M.L.: J.Immuno1.89: 782 and 790(1962)
14. Bauer,D.C., and Stnvitsky: Proc.Nat.Acad.Sci. 47 : 1667
(1961)
15. thr,J.W. and Finkelstein,M.S. :..J.Exp.Med. 117 : 457 (1963)
16. Flodin,P. and Killander,J.: Biochim.Biophys,Acta 63 403
(1962)
17. Dulbecco,R., Vogt,M., and Strickland,A.G.R.: Virology
2 : 162 (1956).
- 18. Iospalluto,J.et a1.: .j.Clin.Invest. 41 : 1415.(1962)
?19. Bauer,D.C.,-.Mathies,M.j.laand.Stavitsky,A.B.: J.Exp.Med.
117_: 889-(1963)
!. I
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-- r?,111
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g
? VUI VntAl'au
?. Table .1
Coll strains, Coll' strains
-in S-form. in R-form
378 346 055 322 16 3A 288
? Unstability in suspension
, heated 100?C - 1 hr
ONO ONO
+
Agglutination in acriflavine
solution 1 : 500
4Mi Ole
Electrophoretic migration
velocity mm/3 min.
24 20 20 61 55 62 80
Phagocytosia: -
% of bacteria. removed
by liver perfusior.
11 16. 30 14 64 55 89
Dilution of precolostral'
pig1et .
serum for 50%
A precolostral
bactericidal
effect calf
0 ?11128 1:128 1:8
????? .0???? ????????? mi????? ????? ?.?? ?????? ??MID ????
OMINOW
1:256 1:2'56 1:64
1:1 1:2 1:1
?.;
?
c.itIC.1
. ? ? I ?
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Declassified in Part - Sanitized Copy Approved for Release 2014/01/15: CIA-RDP80-00247A004200090001-4
a .?
?
: Table 2
Dependence of bactericidn1 activity of piglet sera contain-
ing different amounts of complement on the surface character
? of bacterial strains
, 1
C H50 units/ml ,
of piglet sera
S
R-form
coli 16
-form ---------
coli 127
coli 346 ,.coli 378, Shig.ccli3A
_____.,.
1
0
0 ; 0 11:1
1:8
1:8
1,5
S a
-
o o 1:1
1:8
1:16
2,2
0
0 0
1:2
1:8
1:16
3,1
0
0 0
1:4
1:32
1:32
5
0
1:1 ; 1:1
1:6
1:64.
1:64
Table 3
Demonstration of antibodies to Shigella by different methods
re
?
?
reaction
dilution of serum
1 : 1280
cnti-
Shigella
rabbit
serum
bacterial agglutination
passive hemagglutination 0,1%
650.000
passive
hemolysis
0,25%
0,1 W
0,001. %
65.000
280.000 '
1,500.00.0
bactericidal reaction
newborn
calf
sera
500.000
bactericidal titer
32, 16,
passive hemagglutination 0,1% 0
passive hemolysis 0,001 %
newborn
piglet
sera
bactericidal titer
passive hemagglutination 0,1%
passive hemolysis 0,001 %
1, 2, 1, 1,1 ...
0
_
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Table 4
Uptake of E.coli 346 .by the perfused rat liver suspended
in Ringer-Locke and after opsonization by complement of '
, newborn colostrum free piglets
? -
Experimental conditi
Total number
of experiment
of
% of bacterial
average
uptake
maximo&minim
values
'
0,1 ml. E.coli 346 + 14
0,9 ml. Ringer-Locke
10
(28 - 0)
,
0,1 ml. E.coli +
0,9 ml. native piglet ,
? serum (p.s.)
-
48
.(63 - 30)
0,1 ml. E.coli + .
019 ml. p.s. Paact.30 min,- 5
? at 560'
,
9
(24 - 3T
0,1 ml. E.coli V
0,9 ml. p.s. + EDTA
12
(21 - 5)
0,1 ml. E.coli +
0,9 ml. p?s. absorbed by
zymosan
V
.
10
(18 - 0)
0,1 ml. E. coli +
0,9 ml. pos.treated with NH3L 3
V
`12
(23 - 2)
,
- ?
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?
? .
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-
V.
Table 5
Antibody formation in precolOstral sterile piglets immunized
; with difterent antigens immediately after birth
? The type and amount
? of antigen_
,
Days of life
5 7 10
Salmonella paratyphi B
0
0
0
1:1
/
9
2,5 x 10 heat inact.
0
0 -
1:1
"microbes
0
0
0
2
0
0
'8
1:1
10
.
.
Sheep red blood
0
0
16
256
10 ml
cells 20 w/v .
0
. 2.
16
64
.0
4
16
256
20 ml
0
- 2
16
64
107
T 2 phage
40
320
320
particles 109
0
80
640
- 1280
/ 50 % bactericidal rection with S. paratyphi B estimated
at 3 hours after incubation at 37?C
Hemagglutination with inactivated sera (for 30 min. at
56?C) with 0,1 % suspension of sheep red cells
/ 50% inhibition ,test in the presence of precolostra1
calf serum ,complement diluted 1 : 5
?
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'Tablo; 6
omparison of formation.of,antibodies to sheep erythrocytes
in'preco1C8tra and colostrum-fed piglets
i
20% suspension
Days after immunization
of sheep
erythrocytes
0
P
3
5
7
-
10 ml
0
0
0
1:16
1 : 256
Pre-
10 ml
?
0
1:16
1:16
1:256
, colost- 20 ml
ral 20 ml
0
0
0
0
0
1:2
1:16
1:16
1:64
1:64
10 ml
1:16
1:16
1:8
1:16
1:32
ColostI 10 ml
1:128
1:8
1:3
1:16
1:8
rum- 20 ml
1:32
1:16
1:8
1:8
1:8
fed 20 ml
1:32
1:16
1:16
1:8
1:8
. - Table 7
Primary and secondary response tO sheep red Cells in newborn
piglets
Primary
Secohdary ?
age in
days
serum
serum treated
. .with
0 05.M
ME
! 1M
1 age in
days
1 ' ?________1_005M1M
.
.serum
-...?--------
serum treated
with
ME
- -
....._
1'
RBC 0
?
-
-
! RBC ?
0
-
-
_____B5
'
-
1* . __L...3.5
1
, . 0
9
2 .
-
-
,
9
16
. 0
0
. .10
2
-
-
10
?
1024
512
0
11.
4
-
11
8192
2048
0
13
8
-
. 13
8192
1024
0
15
128
0
'15
8192
2048
0
'17
256
0.
17
-4096
512
8
.. ...19 ,
128
.0
?
0
19
2048
-
-
21
256
.
? .21
4096
?-
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?
Table 8
Effect of complement.on 50 % phage'neu-lralization
?
?
.
days . after
immuniz,
with 10!
T2
serum dilution
1:5
1:10
?
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
1.:256O
compl.
1:5
added
5
11%
15%
28%,
34%
82%
10 ?
-14%
17%
15%
22%
28%
38%
50%
1
67%
67%
71%
15.
6% ?
17%
,20%
21%
20%
22%
42%
...rm???
62%
82%
98%
heat
inact.
compl.
1:5
5
33%
75%
10
30%
58%,69%
103%
15
33%
?69%
112%
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?
Thb,Inductive Phase of Antibody Formation Studied with
'Isolated Cells
-IfeselST/
Jilek, ?
? ?
and Mandel,
Institute:.df Microbiology, Czechoslovak Academy of Sciences,
:
Prague, Czeohoslovai!cia
At the. Symposium in 1959 we summed up experithental-results
..which made it possible to reach the Conclusion that the first
*phase of ,antibody formation can be considere'd to be functionally
distinct /processes -talcing place dUring that phase/ from the
later phase - the actual production of antibedies /15/. This
conclusion was based on observations that the initial phase of
\
-antibody foriation is much moro Sensitive to certain 'forms of
..interference /x-irradiation _/61 23/, vitamin deficiency /1/,
the action of 'hormones /3/,. and particularly on data obtained
experiments, with the transfer of isolated spleen cells to
young homologous recipients. /13/./. We showed that if spleen
; _cells are.cultivated with the antigen:in Vitro antibody format
does not occur; cella' potentially capable of antibody format-
,
',ionjimmunologically competent/ only survive in tissue culture
.
and., the proces of differentiation for antibody formation starts
only when they are transferred from tissue Culture to tissue
.culture in-vivo in:a.newborn recipient /14/. Using this method
'of transfer we showed that the smallest number of spleen cells
?
capable of forminea sufficient level of.,antibodies-in young
reciPients.,for deted-fton bY trio agglutination'reaction /i.e.
about 0.1 ,ug N antibody/mi./ is 106. We further found that
of this nuMber probably only in the order of 103 cells participate
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rr Fth
Cr91%qc'r ONLY
'A A V ugs.
herzl et al. - 2
in actual production, i.e. about 0.1%.. On the basis of these-
? quantitative findings we were able to exclude the simplest version '
of the clonal selection theory,-by which antigen acts directly
by the proliferation of cells already producing the given type
of antibody. Our calculations showed that on this assumption and
:with our methods of detecting antibodies, they should have been
. detectable sooner than they actually were experimentally- /20/.
/.
This was the basis which led to our arriving at the conclusion
that actual production is preceded by a qualitatively different,
Inductive phase of antibody formation whose basis is a change
in biochemical and morphological properties during the process
of differentiation /W.
In the most recent years we have made further observations
in-stpport of these conclusions. With a model of transfer of
isolated cell 6 we showed that the duration of the inductive Phase
.can only be little s4ortened but that it cannot be eliminated
/17/ if an increasing number of spleen cells is used for transfer.
. We used a whole, spectrum of inhibitors of nucleic acids and found
. that only a few inhibit, antibody formation even if those which
.do not affect antibody formation have marked antimitotic activity
,/18/. The. nucleic acid inhibitors, e.g, 6-mercaptopurine and 6-
,, thioguaninel.act only during.the inductive phase of antibody for-
.mation./16/. On a, similar model, the transfer of spleen cells to
:lethally irradiated isologous recipients', Makinodan et'al. /9/
reached the same conclusion, i.e. that the first phase of antibody
formation /the latent phase/ actually exists and can be explained
in terms of disorganization:and?reorganization of the germinal
centi4e.
There are however, certain data, obtained for the most part
IVOR Crik
E11
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herzl 'et al. - 3
)with phage and virus Antigens /12, 241 5/, which are at variance
, .
with these results and conclusions. These authors found increas-
ing titres of antibodies In the serum very soon after giving
antigen and by the interpolation of the results almost elimitated
the.possibility.of the existence of a period during which antigens
were not formed. However, since,in our experiments, particularly
with the immunization of young animals', we continue to reach
the opposite conclusion /19/ we attempt in the experiments sub-
mitted to provide further evidencp.
Direct proof of the existence of an inductive phase would
be provided if, using a population of a sufficient number of
cells /i.e. at least 107 108/ and a sensitive method, we could
demonstrate directly on the cellular level that antibody produc-
ing cells do not appear?.for a Short period after the administrat-
ion of antigen, or if such cells are 'already esent in a given
number, that this number does not increase during a short period
after antigen injection. We selected the plaque technique of
determining antibodies which was introduced by Jorne/8/ as most
suitable in resolving our.problem-and modified it by using agarose.,
a substance forming a gel medium without an anticomplementary
effect /4/. The test was further modified to permit morphological
and autoradiographic observations of antibody producing cells
/22/.
Method: Cells isolated from the spleen or lymph nodes were
washed three times and diluted to a given concentration in Parker
solution with 0.5% HSA,and'immediately before mixing temperated
, .
at 42?C. One part or cells was added to two parts Of aiarose
-containing washed sheep erythrocytes /3 parts 1% agarose in
Parker solution with HSA + 1 part 6% erythrocytes in Parker
solution with HSA/ which was also temperated to 42?C, The mix-
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herZ1 et al. - 4
:ture of cells, erythrocytes and agarose was pipetted drop by,
drop from a height of 'about 60 Cm. into a Petri dish or onto
,a elide. Drops with a- didmeterfof 18 Mm. and single layers of
erythrOcYteS and lymphhtic cells are formed. If the drop is dried.
it'has a,thickness of about 20,u. The drops are incubated at
37?C in a moist atmosphere with 5% carbon dioxide for 6'- 20
hours. After incubation drops are overlayered with guinea ,pig
complement absorbed with. sheep 'erythrocytes. One ml. of comple-
ment used contained 20 units. The plaques are counted after in-
cubation at 37?C for 1/2 hour, and the complement then removed
by veronal buffer and the drops fixed in formalin vapour. After
fixation they are .washed in distilled water and dried. The pre,-
parations were stained ivith Giemsa-Romanowsky diluted 1 20
'for 3 - 5,min. for the morphological determination of the lympha-.
?tic cells. For autoradiographic determinations they were covered
with stripping film Kodak AR10.
We first determined the dyndmics of the increase in the
number of antibody producing cells after i.v. immunization of
mice of a noninbred I strain/weight approximately 20 g./ With
0,5 ml. 1% suspension of sheep erythrocytes. 'Before the addition
of antigen we find an average of 65 antibody forming cells froth
the total number of 108 cells /minimaI.10,, maximum 140/; 24 hours
after the addition of antigen we find, on an average, double the
number. This increase, however, is only found in some individuals;
in others the number remains within the limits of the 'initial
value after 24 hours and may even approximate to the minimal
number of cells determined before immunization /17 to 10 Cells/
108/. In Other animas after 24 hours we found the highest values
;which were found in non-immunized animals /110, 129 159/108/,
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11,? ? ?/
).
herzl . et al.'- 5
? .. ,
? : ? 4
in sone, 'however,.the.numberwas.double he ?
beforeH_immunization,/577 515/10$;... A :uniform increase in the
number of antibody-producing cells an average of '1,741 occurs
Only After 48 hours and. the highest values are 'obtained after
,". .
4 and.5 days and after that the-number of cells producing haemato7
lytic,antibodieS in the, spleen decreases. Since, the largest number
,
of cellS forming haematolytic antibody was found on the fourth
day we investigated the MOrphological characteristics of antibody
producing. cells at this time, The cells were cIaaried accord-'
? ing to the usual convention /10/ and we found that 25% of anti-
body forming cells were small lymphocytes, 47% were medium-sized
'lymphocytes and 27.50arge lymphocytes. Cells which would
-probably gradually be: transformed into typical plasmocytes were
seen only rarely.' The greatest increase in .the number of antibody
'producing cells was between thelthird and fourth day; we wished ,
? to determine whether the increase in'tl'e coil population at this
;
time was due' -mainly to mitotc activity. It would alsci be possible
? .
',for the quantitative increase to.bc due to unequal oontaCt
wit4 antigen -and variations in the,time.dUring which cells develop
into antibody producing' cells: under the:inluence of antigen. If
.the first theory were correct' most of, the;cells would incorporate
labeled thymidine between the third and fourth day. We, first
,
used thymidine-H3 but it 'was found that.most.Of the cells remain
:deeper that 2'u-below the Surface in thethin., layer--of agarose
1 '
so -that- labeled cells were not-detected. We Were more success-
thymidine-0-4 whose effect penetrates to a. distance
of 90;p. /2/. In th'e first cueriment thymidine-H3 in 'amounts
of 1.0 10, Was injected 1.13: ,into -Mice three times at' intervals Of
6: hour's, between the third and-fourth day after immunization. Only, ,
/./
.10% of the centric' /antibody producing/ celle in the plaques,
?
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herzl et al. - 6
were labeled with thymidine. Ir the subsequent experiment we there-
fore administered thymidine from.the injection of. antigen every 8
'hours to 72 hours., i.e.a total of 10 doses /one mouse received
a. total of '50C/. In these experiments we found that the total
number of labeled small, medium-Sized and large lymphocytes was
25%, the large lymphocytes being labeled 100%. The same,perden-,
tage, i.e. 25% was labeled in smears and in agarose, if the drops
Hwerer fixed immediately without incubation. Of a total of 50
examined centric cells only 40% had incorporated thymidine. If
We add 10% of cells which could have acquired labelling between
the third and fourth day, during which thymidine was not given
.in this experiment /on the basis Of the preceding experiment/,
We consider it to be definitely established that not all cells
detected as antibody producing cells, arise by mitotic division.
? lThe experiments determining the time course of the increase in he
number of antibody producing cells in mice make it clear that
we are dealinz'with a most heterogeneous population with a.diVerse
individual history. In nonimmuniZed adult Mice there is not only
. variation in the number of cells in different Aindividuals before
'immunization, but after giving antigen the rate of onset of
antibody formation which 1.6 very marked already after 24 hours,
shows individual differences. We consider that the individual
heterogeneity is the result Of the diverse immunization history
in adult animals and :tt is probably the reason for the diverse
, results obtained.when' immunizing adult animals.
On the other hand our ,conclusions on the time course of
the Onset .of antibody formation are mainly based on developmental
,. studies during ontogenesis, We have repeatedly confirmed /21/
that areal primary reaction can be expected with most antigens
only in newborn aniMals. We therefOre determined the number of
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terZ1 et
antibody forming cells in.,newborn rabbits'from the first to the
50th day of life, both in nonimmunized animals and' in newborn
animals injected i.p, at different ages with 1 nil, 10% sheep
erythrocytes,. Shortly after birth we did not find a single
cell in lymphAtic tissue which formed antibodies. Only from
the 20th day did we find very small numbers of cells producing
antibody to sheep erythrocytes /Tab.2/ in nonimmunized rabbits.
On' the Other hand, as found previously by Aiha /11/, if sheep
erythrocytes are injected into newborn rabbits, antibodies are
.already formed on about the fifth day of life. In accord with
this, antibody producing cells are detected at this time by the
plaque method /Fig.3/. It is also evident from the experiments
on rabbits that the number of cells detected at-the same hours
?-after antigen injection increases continuously with age..Whether
this was due to the spontaneous maturation of lymphatic tissue
or be antigens of the. intestinal Microflora or Mod antigens
which have chemical groups in Common with the antigens of sheep
erythrocytes /7/, we aitempted to decide by further experiments.
'To solve this question we used once again the model of
sterile piglets Which Imre fed. on a honantigenic diet,' as
described previously. If we determine the appearance of producing
cells in normally reared piglets /Which received colostrum and
were reared with the mother under.normal conditions/ we do not
find antibody forming cells' immediately after birth or on the
seventh day of life, but already on the 14th day there are 9
antibody forming cells per 108 spleen Cells,/i.e. 65 cells in
the whole spleen/, .in another animal of the same age .we found
-277 cells calculated to the whole weight of the spleen, These
results stand in sharp: Contrast with those obtained in piglets
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herzl et al. - 8
reared under'sterile conditiOns on a nonantigenic diet, Up to
one month:, i.e.* for the whole period sterile-artificially fed
piglets were reared we did not 'detect one antibody-forming cell
to sheep erythrocytes if antigen had not been given. On the other
hand, if the 'piglets are given an injection of sheep erythrocytes
110 ml. 20% sUspension.of shoop erythrocytes on the first
day of life, the first antibodies can be detected after 72 hours
Fig.4/. Whether the number of cells detected at first
./72 hours after immunizationichanges with age is difficult to
decide on aCcount of the small number of results': It would seem,
however, that it is more Affected by individu?.1 factors than
:by maturation, as we thought originally /18/.
The resultsshow explicitly, that antibody formation does
'not start spontaneousl during individual development if the
individual is protected from antigenic. stimuli. The proof that the
so-called "spontaneous" development of antibody forming cells
in normally reared piglets is due to antigens encountered by the
,animal, appears tb. be given by the experiment in which the number
of antibody forming cells was determined in sterile piglets fed
oh an antigenic diet- degraded cew 's milk, On the 30th day of
.life 7 plaques wore detected in one animal and'10 in another,
In both cases, however, not a single antibody forming cell was
present,in'the lymph nodes /Tab. 4/, The difference between the
pnset of .antibody formation in normally reared rabbits and sterile
piglets /Fig.5/ is Probably that in the first ea-6 antigen acts
development while in the ,second andantigenic stimulus
Is not present.
However, there are several conclusions that Are common both
for rabbits and sterile piglets.^On the basis of the data obtained
in 8 - 10-day rabbits it can be excluded that a previously exist-
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FM OFFICIAL USE ONLY fterzi et al. 9
ing'eell in the 'organism at thetime of immunization Could give
,ripe by proliferation to the number of cells determined after -
the negative'period. If We assumed that the number of producing
:coils arose by proliferatiOn, then at zero time in the rabbit
there would have to be only 1/250.antibodY producing cell for
8
the total 10 spleen. cells. One antibody-forming cell would
only be present in about 1011 lymphatic dells, which is much
more than the young organism actUally contains /this amount? '
.would correspond to about 10 - 100 kg. rabbit/. The situation'
in pigletS is similar to that in infant rabbit. ,In this case
the onset of antibody formation is a little 1-4ter and therefore
in the ze-ro hour, only 1/1000 of a cell from the entire populat?
ion of 108 lymphatic cells wouldlDe able to take part in anti-
body formation. This would correspond to the presence of one
cell in 1011 lymphatic cells, thus again a number' greater than
that present in a newborn piglet. The second possibility that
at zero hour one cell is present which reaches the number deter-
mined in 72 hour's by the very slow process of proliferation in
.untenable since at 24 and 48 hours the producing cells could
actually be detected in, both models /piglets and newborn rabbits/c
If an 'estimation is made of the doubling time in both models
the basis of experimental data obtained between 72 .and 96 hours,
:it comes to 5 hours. On the basis of the above calculation and
the very short doubling timc we are of the opinion that producing
:cells do not arise by a process of proliferation of preformed olis
,
It would certainly,be correct if we could provide direct proof .
in the newborn that the. production cell which isfirst detected
' does not arise by division and does not incorporatethymidine-C14'.
We have no such proof at present for technical, reasons In order
ki71 11SE MY
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terzl et al...7 lo
to detect .tho;very small number of cells appearing in the
newborn it ,is necessary to use concentrated cell, suspension;
106 cells :per,1:m1..'?,If ;cells are.'addecin this concentration
to agarose, the area of 't1.1e. plaque . of 0 1 mm.. Contains appro-
ximately 4,000 lymphatic cells. it is therefore impossible
' in such a concentration to estimate .what cells:are centric)
'i,E3, prQduCtive.
The second more probdble conolusion.which'is also in
?keeping with the preViout findings with the isolated cell
transfer method assumes that for, a certain time after antigen
. .
'injection during the ' primary reaction the Pell passes through the
inductive phaseand does not .produce antibodies and tbat process-
-es. take place during .this time which dre distinct from the later,
'process .of actual antibody production, It is potsible that part
of the Cells which .are capable of responding to antigen /cOm.,..
,
-petent cells/ .divide :already in the course of functional trans-
.formation.when antibody is not yet produced. However;, on the
basis of .experiments with the incorporation of thymidine into
antibody producing cells it mutt be accepted that at least a
part of competent cells is 'transformed into producing cells
'without mitotic division.
;We will attempt to treat the experimental results &n the
light of the. basic unresolved questions of the origin of anti-
'bodies, If we consider the thebry of the existence of multi-
potent stem cells capable of reacting with different' antigens,. '
:.then at. tbe time' of adirlinitration of the antigen only .an un-
- .
probably .small amount of stem cells would be present in a
)1 ? -
"'physiological. condition .allowing differentiation according to
? .
'the type of stimulus.-,If we. find that out of a. total of 107
lymphatic cells ? only- one cell mould be just in the Physiological
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terzl et al..... 11
_state permitting t' to react to any antigen, then this hypothesl:s
e
.seems highly unprobable'. In adult animals the ratio of the tofal
number of cells to the producing cells shifts to the side of the
producing cell. In our experiments, we have 'hown that this
.occurs under the influence of antigen'. It cannot, therefore,
be doubtedthat there is. .a certain form of branching process
.in cell population capable of responding to a Corresponding
I ve
antigen. It would seem, therefore; that the quantittibi results
obtained in newborn developing animals point to the hypothesis
of the selection type in which antigen would act either on
certain cells genetically preformed or arising by a mutation
process and selected by the antigen. These immunolOgically
competent cells, diff6rentiate biochemically and morphologically' '
'during the tnductive phase4finto antibody'prodqcing cells.
References
1. Axelrod, and Pruzansky, Ann.N.Y.Acad.Sci. 63: 202
71955/,
2. ' Baserga, R., and Lisco, J4Nat;Cancer Inst. 31:1559 /1963/.
-)
3. .Bergiund, K. and Fagraeus, A.: In Atti VI.Congr.Intern.
,Microbiol. Roma 1953, vol.2, p.231.
Bernovskal J.,,Kostka, J., and herzl, Polia.microbiol.
9:..376 /1963/.
' Bradley, S.G. and Watson, D.W.: J.Immunol. 901 782 /1962/.
Talmage,. D.W. and Maurer, P.H.I J.Immunol. 68:
,
693 /1952/.
Jenkin: In Advances. in Immunol. 3: 351 /1963/.
Jerne, N K.'. and Nordin A.-A.1 Science 140: 405 /1963/.
sTerne, N.K., NOrdin, Henry,.C.I' In Cell bound
antibodies, Philadelphia 1963.
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?
?
t;a1. 12,
?.
Makinodan, In IIIrd Intern.Symp.ImMunopathologY,,
,Basel 1963.;
Maximaw, A.A. and Bloom Textbook of histology, 'Baltimore
, .
-
U. Iha I.: Falia-micrabi61. 81 1/1963/. .
12. Svehag, S.E. and. Mandel; B.; Virology i8:508 /1962/,
Svehag, S.E.
13. 'tterz1;-J. r Fol.ta biol. 1:'.193 /1955/.
tterzl,, J.: Folia biol. ;3: 1 /1957/.
tterzl, J.1 Folia miarolhol. 4.: 91 /1959/.
15. tterzl, J.: In , SyMp.Mechanisms of Antibody Formation, '
;. Prague 1960,. p.107.
tterzl; Nature 185: 256 /1960/; -
6terz1,J0 Fol.microbiol. 5: .364 /1960/.
?tterzl, Ji: The inductive phase of antibody formation,
State Health Publ.House; Praha 1960;
terzl, J. and Trnka, Czechosl.Epid.Microbial.Immunol.
10: 14811961/. t ?
. 18. tterzl, J.1 Nature 189: 1022 /1961/.
and Mandel, B.: J.Exp.Med. 119: 1, /1964/.
.19. - tterzl, j.; J?Hyg..Epid.Microbiol.Immunal.:7: .301./1963/.
.20. '.tterzl, J. and Trnka, 405./1959
, 21. ?: tterzl, . J. ,and Miler, 1, Folia MierObiol. 6:
,289 /1961/.
- ?
22. ? tterzl, and Manael, L.: Folia Microbial. 91173 /1964/..
. .
23. Taliaferro,' W.H., Taliaferro and Janssen, -E.T.: J.
? Inf.Dis., 91: 105/1952/..,
Uhrl; Finkelsein, M.S., and Baumann, J.B.: J.-Exp.Med.
115: 655 /1962,4.
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F;
Table. 1.
Number of.siitibodY''prOduciilg cells in individual mice
/ . .
.SE'Oep :eryth'rocytes.
! -?
:Days _ I4umber, of plaques per i08
after spleen cells
. limmu-
nization .1
Non-
immunize0,10, ao, 23, 23,, 0, 0
? ' 107, 140 ?
1 day'
Average -
-number
Eof anti-
? .body 'pro- ?
:4Uding
:Cells n
per 10? ?, 1
? spleen :cells:
Percentage.
of antibodY
producing.
cols per
10? spleen:
cells
55 0,00005
10, 17, 110t 1291 159, 315, 377j 159
2 days 356,1237, 25304 2843
I-
,
, 3 ,days 2131, 5747, 7833,9750
'1 4 days, 121300, 32600. 35500,_ 46200
5 days 116000,
9667,
10050,
' 7 days
8 days.'
9 days
25976, 38192
16580
12870
1741
0,00016
0,0017
,
6.115 0 0061
' 33900 - 0,034
26723 .
r13 084-
11 460.
4920, 16117
10 days 4557t 8687
, 11 days. 3617
? ?
13 days I 833, 3793'
<
r - ?
?: ? L.. ? ? ?
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10 519
0,026
0,013
0,011
0,01
6 622 0 0066
3 617
2 313
0,0036
0,0023
? .
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:
,
,
s.? -
'
'Number of
?( ? .
in newborn rabbits .almmUnized? with 'sheep erythrocYt9s
,
.Table
? A
antibody producing cells /pe 10? spleen cells/
.:Age of . Non-
rabbite immu .
at the . .nized , 24 hrs. : 48 hrs. . 72 hr. ? 96 .hrs . 120 hrs.
time of
tmmunizat=
ion
days.
After immunization ?
Lo o
6
0 234
203
? 192
8
A
00: 1773
9
0
335
10
!
-.0 4 . 11
11
0
? 12
0
' 13
0 0 444
16
0
20
8
26,
3:36, 302
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? "Table 3 ?
. .
. ;Number of antibody producing cells /per "108' spleen cells/
Hin newborn precolotral sterile piglets reared on?non-anti-
Age-of
!piglets Non- ' .. After immunization
at the immunized' . ' ?
tilfie ? --.' 48.hrs. 72 hrs. 96 hrs
,of.immuni-.
120 hrsi,
days
4
,
,
i
:
0,0
0, 0,0,37
37,50
181797,1450 719
6
.
i
1
' 6,53
A
!
?
,
. ,
,
,
13
.0
,
283
.
17
'
0
.
,
18
0
16
,
N.
19
'
? .
010,
0,
0;
0
,
?
29
0.
2..
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Table , 4 ?
Number. of Antibody . Producing Cells in One-month. Old
, Piglets reared Sterile and Ped on Conventional Diet -
.
'
'
._ ,
:weight
in g ,
total
number
of cells
total
i number
plaques
,
percentage of
productive
cells
?
Piglet 3
' spleen .
lymph. node
,
5 17
)
3,29
,
'
6,1 x 108
1,5, x 100
10'
o
,
0,00000164
o ? ? .
Piglet 5
. '
,
.
spleen
-
lymph node
7,43
3,07
?
9,5 x l?
-2,34x 108
7
. .
,
0,00000073
Ol
?
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t
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' t y.
t,?
. . ? .
, TIP! ?11 I
Ut L,
4 ,
On the Rol of. Charge ',and !Optical Configuration in -Antigenicit7
, ? ?
' M. S'el a and S Fuchs
Section of Chemical Immunology, The WeizmanA. Institute. of
-
- ! Science RehoVoth,'--
.,;
I
, The availabil1t7 of Synthetic ,polyipeptide antigens
2,- Zrpermit, a systematic inquiry. into the structural
..
e
basis of; antigenicity. KnoWing the ,Chemistry Of these compounds
it is possible, through a ,study of coiDolymers showing only.,
/
) limited variations in. their chemical .formaulae, to arrive at
; conclusions concerning the role of. various structural
in their. antigenic function. 'From earlier studies. /4/ it was
features
concluded. that the immunogenically important: area of the mole-
;
cule must' 1De accessible to the Site of ' the biosynthesis of
?
the antibody, that the overall shape Of the molecule does not
?
:seem to be an important factor in determining immunogenidity,
and that synthetic 'materials with molecular weights as low as
' 4,000 maybe goodimmunogens. ? Th'rough appropriate chemic al modi,,
I ? ? ?
. fication ? ,non-,antigenic macromolecules maybe converted into
antigens,: while antigerlic ones may became non7antigehic. The
polypeptides investigated contain determinants of well-defined
;J and rather narrow specificity /5/.
.;
:to elucidate whether the presence of electrical
,In, this paper we describe and discuss experiments planned
charges, on a -
;macromolecule is a minimal, requirement necessary to endow it
:with immunogen fC properties, and whether ,samino acids of the,
, ? D-optical configuration -6 which 'normally are not present .in
proteins - influence the immunolOgical response. , Thus, we
inquired whether the attachment of peptides containing -tyro.-
_ ?
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? ? .
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. ,
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.Sine to a poor angen-gelatin --may enhance its antigenicity
-and'Whether.a:siMlar attachment -to a non-antigenic matro
molecule-multichain poly-DL-alanine - may convert it into an
=
-
immunogen. The.study was extended to macromolecules composed
,eXclusively of 1).4mino acids.
Experimental
'Gelatin /Gel/, U.S P..granUlar, was obtained from Fischer
Scientific Co., 'Pittsburgh, Pa. Poly-L-tyrOsyl gelatin /p-L-Tyr
-6e1/0 sample 240, and poly-L-tyrosyl gelatin /p-D-TyrGe1/0
sample 246, were prepared acCording to.Arnon and Sola /6/ from
.gelatin and tile respective N-carboxytyrosine anhydrides. The
chemical characterization 'of the two polytyrosyl gelatins is
given in Table 1,
The nomenolature used in this paper lor linear and multi-
chain copolymers- of -aMino 'acids is that described previously
/4/0 except that the latter defining the optical configuration
of an amino acid residue precedes thd abbreviation of the amino
acid. The linear cOpolymerEL42,p/L.,Tyr,L-Glu,L-Ala/, and
1020p/L-Tyr,L-G/,, were described.in a previous paper /4/..
The other copolymers ligted in. Tables 2 and 3*were prepared
in complete analogy with similar polymers described by Sola
, -et.al. /4/. .
,Tho sedimentation and diffusion coefficients of some copo-
lymers Investigated are given in Table 4. For calculation of the
averageTmolecular Weights,' the partial specific volumes of the
,
copolymers were computed from the partical specific volume of
the 'amino acid. residues and their proportion by weight in the
oiymor /4/:
A mUltichain cepolyMer was prepared in which peptides of
?
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L-tyrosine, and N -/3hydroxypropy1/-11-:s1ubam1ne are attached
to ?multichain poly-M-alanine. The designation of this substance
is p/TyroHpe-pAlaTpLys. Tt was synthetized as follOwst
? Multichain poly-DL-alanine /pAla.pLya/ was .reacted as usual /4/
=
with the N-carboxyanhydrides of L.-tyrosine and benzyl-L-gluta-
? Tnate. The product.was reacted with propanolamineo according to
Lupu.et al. /8/. Under these conditions the benzylglttamate
5 ?
residues were converted, quantitatively ? to N -/3-hydroxypropyl/-
glutaminyl residues /Hpg residues/, according to the following
'scheme:
CH/CH / C0000H C H + NH /CH / OH
- / 22 2 6 5 2 23
-NH
CH/CH / CONH/CH / 0H+C H CH OH
22 23 65,2
The resulting 218,P/tyr,Hg/-pA1a--pLys, had a tyrosine
awmtant of 12.5% and an average molecular weight of 30,400
caloulatel from a sedimentation coefficient of s200w = 2.1 So
7 2
. a diffusion coefficient of 11200w = 5.6 x 10, cm /s and a partial
specific volume of 0.70..
An uncharged_multichain copolypeptide /designated
dea-p/TyroHpg/-pAla-pLys/ was obtained from the above polyMer.
upon desamination /Fig.1/. The'p/TyroHpg/pAla--pLys /1 g/ was?
-
desaMinated with nitrous acid /500 ml 0.0625 N aodium nitrate
and 120 ml 2.5M acetic acid/ for 1 houb at 37? C. The dialyzed
and lyophilized product contained noamino groups /Van Slyke
analysis/.
Methods of immunization of rabbitAl precipitin and inhibit-
i.on tests as well as physico-chemical measii,uments used in this
,
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study are those:deS;cribed. previously. /4,5/. When Antigens.
werereacted with antisera at different pH values, the follow-
ing procedum. was_used:Ahe antiserum was dialyzed for '48 hours
against two changes of a tris-malonate buffer of the desired
pH and an ionic strength of 0.15 /sodium chloride was added
to 0.05M tris-malenate to adjUst At to a constant ionic
strength/; at the end of this period the contents of the dia-
lysis bag were centrifuged and the supernatant was used in
precipitin tests.
Antigenicity of an unchanged synthetic polypeptide
Electrical .charges .on antigenic determinants have been
assigned a crucial role in defining the antigenic specificity,
affecting the charge distribution on the combining sites of the
antibodies, and contributing in an important way to the forces
Of the specific interaction between an antigen and an antibody.
Singer /9/ has reported that in each antigen-antibody bond
.in several different systems there 1.0 critically .involved a
singre pair of oppositely charged groups. Kabat /10/ has
-questioned the direct role of the charged group in the antibody
combining site, emphasizing that charged groups cannot play
a significant role in the specificity of uncharged carbohydrate
haptens. In a recent paper ,by Wofsy and Singer /11/ it has been
concluded that lysine residues cannot be vital components of
the reactive sites of antibodies to either negatively charged
antigenic determinants ot to a neutral hapten.
Studying-the effect of attachment of peptides cf various
aminn acids upon the immunogenicity of gelatin, we observed
-:.'that while some amino acids - such as tyrosine or tryptophan
?. ?
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enhanded the formation of antibodies, the ionized 'amino acid
'residues /either lysine or glutamic acid/ has no signifcant
enhancing ability /12/ though they exercised considerable
influence-on .the specificity of antigens /6/. This has been
observed also with synthetic polypeptide antigens /4, 5/. On.
the other-hand,' the attachment of mixed peptides of positively
and negatively charged amino. acid residues /lysine and glutamic
acid/ enhanced the antigenicity of gelatin and converted the
non-antigenic multichain poly-DL-alanine /P-DL-Ala-pp-L-Lys/
== ==
Into an immunogen /13/. This is in agreement with the observat-
ion.that, vhile neitherpoly-L-glutamic acid /14/ nor
poly-L-lysine /15/ are immunogenic,. some linear copolymers of
.;,-lysine and L-glUtamic acid possess the capacity to elicit
antibodies /16,17/.
The ciestion,arose whether electrical charges are at all
necessary to endow a macromolecularwith Immunogenic.properties.
Detran and levan are devoid of charges and are Immunogenic
Iii humans /10/. We have endeavoured to prepare a synthetic
polyp'epide that would be water-soluable, non-ionizable and
- immunogenic. A preliminary report of our work has been published
.recently /18/. ;
The synthetic multichian polypeptide antigen, p/L-Tyr,L-Glu/
/1,4/contains both negatively Charged tarbo-
= =
? xylate ions of the 'glutamate residues, and positively charged
-.ammonium ions at the termini of the polymeric side:chains. The
polymer.p-L-Tyr--p-DLyAla--p-L-Lys contains no carboxylate ions,
== = ?
.but the removal of the ammonium ions by desamination with
nitrous acid converts it into a water-insoluble product., In
order to obtain an uncharged water-soluble analog of p/L-Tyr,
L-Glu/--p-DL-Ala--p-L-Lys, multichain poly-DL-alanine
==
7 ==
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Sela - 6
/124.,DL-A1a-.-p-L.Lys/ was reacted as usual with the Y-carbO-
=
? xyanhydrides of L-hyrosind'and benzyl-L-glutatate, but instead
of removing the ,benzyl groups with anhydrous hydroen bromide
. ,
, to yield free carboXylate ions, the bbOnkylgiutamate residues
5
Were reacted with propanolamine to yiei. N -/3-hydroxypropy1/.
glutaminyl residues. The product, p/L-Tyr,L1.1pg/-?-p-DL-Ala--
, = ==
p-L-Lys which contains many hydroxyl groups, is soluble in
water and still contains ammonium ions at the termini of the
polymeric side-chains. This substance is antigenic: in a typical
experiment. 160 /Ug of antibody were precipitated when 100 /ug -
antigen was added to.1 ml antiserum,
The above polymer was desaminated with nitrous acid.
for 1 hour at 37? C., The dialyzed and lyophilized product
contained no amino groups /Van Slyke/. The reaction is illustrated
schematically in Fig.l. The desaminated polymer is devoid of
charged groups. Nevertheless it is water-soluble, due,, to the .
Ihtroduction into the molecule of many hydroxyl groups upon
the reaction with, propanolamine..
The desaminated polymer elicited antibodies /300 lug/mi
serum/ in rabbits immunized in Freund's adjuvant, as checked
byAlomologous precipitin reaction /Fig.2/.. The antiserum cross-
reacted to a smaller extent with the non-desaminated polymer
/which carries some positive charges/, and gave only a poor
cross-reaction with the highly charged 11/t-Tyr0L-Glu/T-p
DL-Ala--p-L-Lys /Fig.2/. The last finding suggests thatno
== ?
conversion of hyaroxypropylglutaminyl to glutamyl residues
had occurred in vivo between the time of injection and the
time of the "imprint" at the biosyntheticsite.
It may be concluded from the above experirents that
a completely uncharged synthetic. polypeptide possessing the nece-
ssary Immunogenic features /in this case tyrosine/ is capable
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Of eliciting antftodieal in rabbits And, therefore, - that the
? presence of charged ..groups in the molecule is not always
essential for immunogenicity.
In view of the fact that a molecule completely devoid
of charge may not only induce the formation of antibodies,
tut also precipitate with _them immunoopecifically, it seems of
/-
interest to elucidate in this case the nature of the forces
between the antigen and the antibody, as electrostatic inter-
-,
actions are obviously excluded. Preliminary exp'eriments /in
collaboration with Mrs.E.Hurwith/0 comparing the pH dependenqe
of the homologous ahd heterologous reactions of the desaminated
polymer ad of p/L-Tyr,f-Glu/--pDL-Ala--p-L-LysAwith antisera
==
to these two antigens are illustrated in Fig.3 and Fig.4.
No significant differences are observed between the two
systems In both cases the amount of precipitate decreases .
when the pH is either raised or lowered from the neutral region.
As the pH could not influence the ionization of the uncharged
antigen, the changes in the prcipitin reaction upon varying the
pH must have been due to intramolecular changes within the
r-gloisulin molecule. One could extrapolate from the above to
conclude that even in the case of charged antigens and their
antibodies, the dependence of the precipitin reaction on the pH
does not necessarily result from changes in the ionization of the
interacting groupings on the antigen or antibody.
Antigenicity of macromolecules containing D-Tyresine and some
L-Amino acids
Differences between-optical isomers of organic compounds may
? be detected by immunological methods /19,20021/. The distinct
aerological specificity of antigen.p.eterminants of different
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optidal configurations has been demonstrated also for optical
-isomers Of amino acidS /2123./ 'The question we'asked our-
selves in the study reported here was not primarily whether -
-
and to what extent - the different optical .isomers of amino
acids contribute to antigenic specificity, but rather to what
extent )they are'capable of Contributing to the Immunogenicity
of a Molecule. The amino-acid tyresie was chosen for these
4 ?
s': experiments-.as it was known that the attachment of peptideS.
, ? . .
.,ofji-tyrosine, or of L-glutamic acid,, enhanced the. immunogeni-
_.
._. , . . .
. .
. ci,ty of gelatin /12/1 and converted- multichain poly-DL-alanine
'from a non-antigen into 'an Immunogenic molecule /3,14/.
. .
. Attachment of peptides'of, D-tyrosine resulted in a
.clefinite.increase in antigenicity as Compared with unmodified
-geiatin /Fig..5/. The homologous system of poly-L-tyrosyl gelatin
'is given in the same figure'fOr ,cordparison, Antibodies to p-L-
TyrGel cross-reactdd'only to a small extent with p-D-TyrGel,
? 0 ? .
,and.the sena situation obtained in the reverse:case?
analogy-with the synthetic imonogen p/L-Tyr1L-Glu,/,
. we have prepared a syntheticstultichain
:polypeptide p/I5-TYr,L-Glu/-.-p-DL7Ala--p-L-Lys and tested
==
,Ipihether it is able to elicit an immunoe response.. In Fig.6
?
the homologous precipitin
DL-Ala-pp-L-Lys is given.
==
reaction-of p/D7Tyr1L-Glu/--p- ?
=
?
Thus the attachment of peptides of
'D-tyrosine and L-glutamic acid
=
alanine into an,.antigenic
dos. of: L-glutamic 'acid alone does not confer immunogenic properties
converted multichain poly-DL-
'
. ==
molecule+. The attachment of penti-
+The finding was 'discussed in a prelimiriary form /24/.
0
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_ ? .. .? ??? ? ? . 4.4,11 .4 ? ?
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\.?
?
-
- 0
on multichain pb1Y-DL-alanine Y4/1. Tile 'cross-reactions bi-' the ?
?
-
-;antisera against p/D.-TyrIL.;Glni--p-DL-Ala--p-L-Lys and against
p/L-Tyil?L-Glu/--p-DL-Ala---p-Ii-Lys with thOantigens containing
. . ., ..-..... ? =
r
.tyrosine of the.opposite-optical configuation are illustrated '
_ , .-.?-
;.-----
; : .? . . . -. . - . . .
inTig.7: The contributionlof the optical configuration to the -
. . .
specificity of these antisera is apparent also from the fad'.
/Fig.7/ that the tntilantum.to p/L-Tyr?L-Glu/--p-DL-Ala--p-L-Lys
..= . ...-=.-- ? .
?'reacts, to,a much greater extent with p/L-TF,L-Glu0L-Ala/ than
' with p/bTyrID7GlurL-Ala/.
= . =
? The antigenicity .of the linear polypeptide p/D-Tyr,L-Glu, -
=
t-Ala/ is shoWn,in Fig.t and compared with that of p/L-Tyr,
?
L-GluIL-4a/ in Fig.-8. The respective cross-reactions are also
= =
giVen in Fig.8. Neither of the two antisera cross-precipitated
.With the polymer composed exclusively of r-amino acids, p/D-Tyr,
;D-Glu,D-Ala/.
??It may be concluded from the above experiments that
introduction of D-tyrosine into macromolecules containing also
to-,amino acids, and which are either poor antigens.or non-anti-
genic, may result 'in an increase of antigenicity or conversion
into, an antige4csimilarqy to the introduction of L-tyrosine.
?
-Immunological studies on mac5pmolecules composed exclusively
of D-amino acids
? In view or the r-eSults.obtained with polymers containing
,both D-tyrosine and-L-amino.acids, it was of interest to enquire
.whether synthetic Pplypetides containing exclUsively D-dmino
_.a.Cids -.and among them D-tyrosine which was shown to be an
?
'immunogenic factor -4, mayelicit an antibody-response in rabbits.
We have prepared several linear and multichain polypetides
,composed exclusively of D-amino acids. One linear polymer, 237,
=
?
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iO4???
Li
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?
Se] - 10
-^
? contained only D-tyrosine and D-glutamic acid4 Two other linear
.F81T,mers, 236 'and 247, contained D-tyrosine, D- glwt ami c acid
and D-alanine. A mtltichein polypeptide was prepared from poly-
D-lysine and N-carboxyanhydrides of D-tyrosine and benzyl-D
glutamate, and converted into 248,p/D-Tyr,D-G1u/--p-D-Lys. All
these polymers are described and characterized in Tables 2,3
and 4. Analogous polymers of the L-series, which were shown
previously to be potenti antigens /4/, are also included in
these tables.
The above fair D-amino acid polymers were tested for their
antigenicity in rabbits. Each polymer was injected into eight
rabbits, in an intensive course of immunization /4 injections,
every 10 days, in complete Freund 's adjuvant followed by '4
Intravenous bobster injections/. The sera were examined with
the homologous polypeptides: and with L-analogs, before each
Injection as well as for three months after the last injection,
._and found to be in all cases completely negative as followed
by precipitin reaction. The sera pooled at the end of the
. immunization period were .checked also by passive cutaneous
anaphylaxix'in.guinea pigs /we are grateful to Dr.., V. Borek
and Miss J.Stup for this experiment/ and found to be nggative
. both with the polymers composed of D-amino acids and those
composed of L-amino acids.'
These results are -ih agreement with these reported recent,
ly by Gill et al. /25/ and by Maurer /26/. Gill et al. have
. found that a. copolymer of D-glutamic acid and D - ly s in e did not
. elicit antibodies in rabbits, even' though and L-copolymer of
a similar composition was immunogenic /25/.' Maurer has report-
' ed the lack of antigenicity of a cOpOlyner of D-glutamic
? acid and D-alanine and of a copolymer of D-tyrosinel
,D-glutamic acid and D-alanine
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Sela - 11
/26/.
In this conneCtic?n it is pertinent that, While an,azo-
? benezenarsonate derivative:of,poly-L-tyrosine induced delayed
hypersensitivity in guinea pigs /27/0 a similar derivative of
poly--,Dtyrosine' was immunologically inert in this species
./Borek, Stup, .and Sola, unPublished'results/,,An azoiSenzene-
arsonate derivative of a copaymer:of D-tyrosine, P-glqamic
acid and D-alanine was also shown recently ?to lack the capa-
=
city to induce an immune response in guinea pigs /28/.
It is apparent that polypeptides composed' exclusively of
fl-amino Acids differ basically from those containing L-amino
acids in their capacity to elicit an immune response. It is not
at all clear whether this difference is due to the lack of pro-
teolytic.enzymes capable of splitting peptides of D-amino acids
/this assumption would imply that at a certain stage of the
ImMunization process such a step is necessary/, or.whether'the
-optical configuration plays a role in a specific fit with a,
? ' complimentary area at some stage of the immunization. A suggest-
1,on has also been made /29/'that the non-antigenicity might be
due to a continuous excess of the undigestable D-antigenr
Gill et al.. /30/ have succeeded recently in eliciting in
. rabbits the formation of aatibodaes against a copolymer. of
D-tyrosine,D-glutamic acid-and,D-lysine. /We wish to thank
,=
L 4
Dr.Gill for the opportunity ;of reading a manuscript of his, prior
to publicatibn/. It seems thus, that some D-amino acid copo-
lymers may be immunogenic in rabbits. It would follow that
:different chemical criteria govern the im6unogenicscapacity of
polypeptides of the L- and D.!.series.. On the other hand, the
-immunological behaviour of polypeptides of opposite optical
configurations might also depend on the genetic make-up of ?
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FORtFil-C'iAL 1:SE.
COncluding remarks
Sela - 12
We have demonstrated tb.gt-a dompletely.uncharged maCro-
molecule a Synthetic protein model Composed of amino acid
residues - possessing the necessary iMmunogenic features may
elicit matibodies_in rabbitsp'and therefore that the presence
of charged groups is. not essential for immunogenicity. '
Peptides containing,D6styrosine possess immunogenic capacity
siMilar to that of peptides containing L-tyrosine, being able
to convert non-antigenic molecules -containing also L-amino acids.
into immunOgens. On the other hand, all efforts to elicit anti-
.'bodies to four preparations of polypeptides composed e7lusively
of :D-amino acids Were unsuccessful until now in our laboratory..
This investigation was suliported.in part by a research
".grant /AI-04715/ from the National Institutes of. Health, U.S.
Bublic Health Se:14,710e.
H2OFFICIL cE ONLY
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i . ,, . .
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.; - ?
-, , Sala -16
,._
.'Referpncps
l'i'Sela; M, and ArnonljZ :. Bioehimet Biophy64Aca 4C:382./1960
2. Gill, III, T434 and4oti, p.t.3.Mo14Bio1,.Z:65 /1961/, .
? .1 ' ? . ? s . ?
'3 Stahmahn, M;At/Editor/: Pblyaminoacids, bolypetides and .
prbteins4 The. University of Wisconsin Press Madison 196,
44 Sefa, M. Fuchs, 5,, and Arnon, R.1 Biochem,J. 85;2230/1962/,
-5.: Fuchs., S. and Se1a_0 Biochem..34 87: 70 /1963/.
6. Arnon, R. and Sela? M.:Biochem.J. 75:103,4960/
.7. EastOe,..J.E.: Biochem.J. 61:589 /1955/,
8. ?LupulN., Yaron, A. .Sela, M. and'Berger,.A.:Bull.Res.Couno,i1
Israel, Sect.A 10:'47 /1961/.
9. Singer, 3,J.: J,6e1lular Compar.Fhysiol. Supplement 1, 50:
,.51 /1957/
Kabat, E.A.: Expprimental:Immunochemistry- Charles C.'ThoMas,
'-Springfielii, Illinois' 1961.
o
.4. Mbfay, h, and Singerl'S.J.: Biochemistry 2:104 /1963/. .
. .
12, Sela,- M. am d Arnon0 Biochem.J..75: 91 /19..60/.
4'13..PUch61 S, and Sala', M.: Biochem.J.,'in press.
14. Maurer, P.H.4.::Proc.Sbc?Exp.Biol.Med. 96:394/1957,)".
e
Subrahmanyam, D., Katchalski, E., and Blout0
'E.R.: J.Immunol. 83:193./1959/. . 1
-..-
r
16. Gill, III, T.J. an Doty, T.1 J,Biol.Chem. 236:2677 /1961/.
K '
17. Maurer, p.H.: J.Immunol. 88:330 /1962/. ;
?
18.,Sela, M, and Fuchs, S.:'Biochim.et Biophys. Acta 74:796 /1963/.
-19.:Lantsteiner, K.: The Specificity of Serological Reations
..Dover Publications, i*c.? New York 1962. '
,
20. Landsteiner, K. and van der'Scheer, 3.: J.Exp.Med:' 50:407;
/1929/,
21. Goebel,
and Avery, O.T.: J.Exp.Med. 50:521,4929/.
22. Ivanovics, G. and Bruckner, V.': Naturwissenschaften 25:250
? .ft . ? ? .
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A
-
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? 0
Sela - 14
/1937/.
23, -Sage; H., Fasman, G., and Levine, L.: Fed.Prod. 22:555 /1963/.
24; Sala, _M., Fuchs, S? anO.'s Givol,p.: Abstracts 143rd Am.Chem,
Ssoc.,Meeting, Cincinnati 9A, 1963,
25, Gill, III., T.J.; and roty, F. Nature 197:
\'76:6 /1963/.
? 26, Maurer, P.H.: Proc.Soc.M45.Biol ed. 113:5E13 /1963/.
. 274 Leskowitz, S.: J.Exp.Med. 117:909 /1963/.
:28,Be.acerf., B. .0j da, A., and Maurer0.P.H.:J.Exp.Med.
118:945 /1963/.
Zul,ay, 6. Nature 200:483 /1963/.
30, Gir:, III, T.J? Kunz, H.W., Gould, H.J., and noty,
J.Biol.Chem., in press.
tr_ ,
?
?
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L.,
-
; Table 1
Polytyrosyl 'gelatins- -
Se's -:15
' Ge.LEitin. derivative Percentage of Percentage of Enrichment bl
tyrosine residue :tyrosine residue B
x 100
in the original in the gelatin 100-C
gelatin a derivative
?
240,p-L-TyrGOL1
0.21,
13.5
15.4
246,p-D-tyrGe1
0.21
13.2
15.0
, From Eastoe /7/.
-1)
Calculated assuming gelatin as 100%.
".`
,
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Sela - 16
1
'Table 2
Linearcopolypeptidesi
,?????????
?
No.ian.e. designation of
-sample - . ?
?
Molar ratio
of N-carl..o-
? xyamino acid
anhydrides in
the polyme-
rization mixture
102,P/L-Tyron--G1u/
237,p/D,.TyrID-G1u/a
42;p/L-TyrIL-..GLuIL-Ala/
? ?
2131p/D-Tyr,L-Q1u1L-Alak
,= =
?2364/D-Tyrop.GlUID.:Ala/
2470/D-TyrIr-GlUID-Ala/
141
1:1
1:5:4
1:5:4
1:5:4
1:5:4
Molar ratio
of amino .
acid residues
?
Weight /%/
of tyrosi-
ne resi-
in the copo-
lymer,
dues in ,
.the copo-
lymer
141
56
1:1.2
52
15.4:3:9
15
1:6:8:3.7
12.4
1:5,8:5.1
, 12.5
13.8
.
. h.
Number average degree.of polymerization Of these samples was
calculated from amip.o nitrogen deteiminations, and found to be
85. and 93 for samples 102 and.2370 respectively. .
??????
? .
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?
OVCICALUMLY
Table 3
Multichairi copolypeptides
Sela - 17
?, ? .
No.and designation of .sample No.and' designation ? . Weight /g/ ,
and volume ?
Ara/ of
buff era
210, p/k- Tyr, L-Glu/-p-DL- Ala
, =
--p-L-Lys
2110p/D-Tyr,L.,.G1u/p-Dt-Ala
==
--p-L-Lys
=,
:249,p/L-Tyr,L-Glu/-- L-Lys
H2480p/D-Tyr,D_d1u/_-p-D-Lys'.-:
?
?
.
208 p -DL -Ala - -p L.Lyse
5?
350
?????
208,p-DL-A1a--p-L-Lysc
2;
140
p-L-Lys
0.7;
70
p-D-Lys
0.7;
70
'
Amount /g/ of Molar ratio of amino acid WeighE-M/ of
IT- carboxyaydrides restdtes in the .copolrner tyrosine re-
of sidues in the,
copolymer
Tyrb
..13enzy1
Glub
Lysb Tyrb Glub DL-Ala
= =
. :1.75
0.7
;0.45
- 2.5 -
? 1.0
2,4
.b.45 2.4
1.65, 2.1 17.2
1.3 1.5 18.3
0.25 1.9
0.28 2,3
OM.
14.3
11.6
9e8
9.8
a
'Phosphate buffer /4/.
1CD- or'L- form
? 0
In a molar ,ration of Lys to Ala
/ of 1:19.4; average molecular
weight 27,800.
,-f011: Of fIG11:114
.? ?
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t
Sela - 18
e.
:Table 4
Physicochenhical properties of some oopolymers.
, No.S.nd designation Of samPle Partial Se di- Diffus- Average
,
specific mentat- ion coef- mnle-
?: -
. volume ion coef-. ficient cular
z _4? ' ? ' fiCient. ' /107 x weight
-
/820,w in
Svedberg
units/
? t?..
4201)/11:-Tyr,L-q1,u,L-Ala/
0.63
213 p/D.- Tyr, L-Glu, L-Ala/ 0.61
236;p/D-Tyr1D-Giu,D-Ala/ - 0.63.
.? = .
247,p/D-Ty'r,D-G111.,D.-A1a/ 0.63
=
210,p/L-Tyr 0.70
z=-1 ?
p-T;e.,Lys
,
0,9
14.4
4,100
1.6
4.65
21,400
1,75
3.4
33,800
1.5
5
19,700
2.25
5.5
.33,200
211 p/D- Tyr, L4Glu -p-LL-Lia-- 0.70 . 2.38 7.3 ? 26,400
= =
p-L-Lys
? ==
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t ?
c-Legend? to Figures.
? ?
? --Fig.1.
Sela - 19 :
,
Schematic presentation of the desaminationreaction
. ? .
, .
leading to thpfteharged synthetic antigen.
'ExtinCiion at 2800 2. of solutions in 0.1N sodium hydro-
xide of precipitates obtained by the'additiori' to, an
:antiserum to the.urIcharged.desaminated polymers,
.0f: 0 0. the desaminated polymer;
p/L-Tyr0L-Hpg/-7p-DL-Ala-.-p-L-LyS; ?
_
p/L-Tyryh-Giu/--p-DL-Ala?--p-L-nys. ?
The pH dependence of the precipitates obtained in the
equivalonce zone .upon reacting antisera to the des-
aminated poiymer, and to p/L-Tyr,L-Glu/--p-J
? = ? =
DL-Alap-L4Lys /-----/, with the desaMinated poly-
mer 7 0 / and'with p/L-.Tyr1L-Giu/--p-DL-Ala-pL-Lys
==
/ 0 /. The antisera were brough to the deSired pH
values as described .in 0.1N -sodium hydroxide and
,--the-extinction of the solution was read at 2800 R.
.Data of Fig.3 normalized .to give .the same maxima].
'precipitation Thr the two homologous. systems. -
the desaminated polymer and its antiserum; -.-.,
?
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p/L=.Tyr,L-Glu/--prDL-Ala--p-L-Lys and the antiserum
? ? ==
to the desaminated .polymer; Ip/L-Tyr,rpr.Glu/--
and Its antiserum, the
qPsRminated polymer,and the antiserum to p/L-Tyr,L-Glu/
--p-DL-Alap-L-Lys4
?
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Sela - 20
o
Extinction at 2800 A of .solutions in 001N sodium hydro-
xide of precipitates obtained by the addition of /left/
p-D-TyrGel:/ 9 / and p-JJ-TyrGel / / to the antiserum
against pAD-TyrGel; /right/ p-L-TyrGel. / ej and
p-D-TyrGel to the antiserum against.p-L-TyrGel..47
'
"Fig. 6.. -. Homologous precipitin curves of 211,p/D-Tyr,L-Glu/--
p-DL-Ala--p-L-T,ys, / 8 / and 213,p/D-Tyr,L-Glu,L-Ala/0
= ,
/ 0 /. The amount of antibody was obtained from the
extinction at 2800 R after deducting the calculated
extl,Action of the antigen. The amount of antigen in
the precipitate was obtained from radioactivity data.
Extinction at 2800 A of solution in 0.1N so.dium'hydro-
xide of precipitates obtained by the addition of:
A - 210,p/L-Tyr,L-Glu/--p-DL-Ala--p-L-Lys, /
211,p/D-Tyr,L-Glu/--p-DL-Ala--p-L-Lys, / 0 /,'42,p
/L-Tyr,L-Glu,L-Ala/, /4V and 213,p/D-Tyr,L-Glu,
= =
'L-Ala/, /6/ to antiserum against p/L-Tyr,L-Glu/--
= =
p-DL-Ala7-p-L-Lys- B; 211,p/D-Tyr0L-G1u/--p-DL-Ala--
p-L-Lys, /0/ and 210,p/L-Tyr,L-G1W--p-DL-Ala--p4-Lys.
== =
Extinctign at 2800 of solutions in 0.1N sodium hydro-
xide of precipitates obtained by the addition of: ,
A; 42,p0p/L-Tyr,p-Glu,L-Ala/, /i4 and 213,p /D-Tyr,
L-Glu,L-Ala/, / 0 / to antiserum against p/L-Tyr,
L-Glu,L-Ala/i B;-213,p/D-Tyr,L-Glu-L-Ala/,./ 0 / and
= =
42,p/L-Tyr,L-Glu,10-Ala/1 /V to antiserum against
=
p/D-Tyr,L-Glu,L-Ala/*
= = .? =
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e -
?TOR pFFICIAL USE UNIT
StUdies on-tile Nature Of ImMunagenity Emploing Soluble
ancIParticulate Bacterial Proteins
I. ?
...L.Ada, G. .V.Ilossai; and Caroline M.Austin
-
- The jolter: and Eliza HalI Institute, Melbourne, Australia-,...-
'Many examples are now knownoin nature of particulate preparat-
,:,ionS, composed wholly or mainly of protein who-se basie structure
ean.be described as an .orderly aggregation of small building
.blocks. A well documented example is.. the protein shell of tobacco
mosaic virus.. The basic Subunit of this structure is a protein of
f.
'low molecular weight., At weakly acid pH, the protein molecules
will aggregate to fOrm hollow cylinders, similar in dimension and
:appearance. to the. Original virus particle from which tlie protein
:Sub-units can be obtained. Other viruses seem to be constructed
-:in a ,comparable fashion and it is becoming clear that this pattern
.,of construction is, widely used in nature. For example, some years.
:ago it was shown 'that the flagella of certain microogganisms
were also built up as linear polymers of small protein units
and this soluble protein was, called, flagellin /1/. It seemed to
us that a situation such as this offeredan opportunity to study
:the antibody response to natural Antigens of similar chemical
properties but of varying size.
? -
Proteins .of both viral or bacterial origin are known to be
..powerful antigens. Bacterial flagella and derivatives made from,
' them were.chosen for.our study, not only for this *reason but
also for their ease. of preparation in A relatively pure state.
Preparation o? three flagella Antigens /2/
Flagella were prepared from cultures of Salmonella adelai4e.
This opganism was chosen as its occurrence in nature is rare and
FOR efFIQ? USE ONLY-
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a ?
Ada et al. 2
high yields of flagella are readily obtained. The flagella
are sheared off the bacterial bodies which are then deposited
from solution' by low speed centrifugation. The flagella are
sedIMented from this solution by centrifugation at higher speeds
and are purified by means of several cycles of differential cen-
trifugation until free low molecular weight protein. Such pre-
parations were used as the first antigen. They are freshly pre-
pared or after storage at -20?C,
Disaggregation of flagella can be made to.occur.in many
ways /3/ but we have used treatment of the preparation with
weak acid /0.05N-HC10 30'mink, 20?C/. Centrifugation of. this
solution at high speed leaves the soluble protein, flagellin,
in the supernatant and deposits about one per cent of the
original flagella as an-acid-insoluble residue. The supernatant
fluid is neutralized with alkali and passed through a sterilizing
filter. The filtrate is clear and colourless. Addition to this
solution of a. concentrated. salt solution causes an Immediate
turbidity. A convenient way to achieve this. is to bring the
solutio to 60 per cent saturation with ammonium sulphate and,--,
after standing, to dialyze the solution against 'distilled water
until salt free, Su6h a solution is opaque,and viscous. It is
termed polymerized flagellin and is used as the second antigen.
The third antigen used in these studies is the soluble protein
flagellin. This can-be prepared as described above directly fi-om
flagella. We,. however, have chosen to prepare it by acid treatment.
of the polymerized flagellin, as flagellin so prepared is less like-
ly to be contaminated with the acid insoluble material present in
the flagella particl9; it seems likely.that.the process Of poly..
merlzation depends upon the specific configuration of the protein
molecule so that the polymerization itself would act as a puri-
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Ada et al. - 3
fication procedure. Such preparations of flagellinl.particular-
ly if the protein concentration is high, will readily repoly-
merize and to minimize the chances of this happening, flagellin
solutions were injedted or studied as soon as possible after
preparation.
Properties of flagellal flagellin .and polymerized flAgellin /2/
Physical and chemical properties. When flagellin is treated
with strong salt solutions, polymerization occurs and the result-
ing polymer, when examined in the el4tron microscope, is seen to
_consist of rod-like stDuctures which are similar _in appearance
. to particles of flagella. Though not examined in great detail,
both preparations consist of rods of varying length and of vary-
ing Curvature.. This variation in size can also be demonstrated by
? studying the sedimentation of either preparation in a sucrose
gradient. Both preparations show a distribution of. Proteins which
extends from the, top to the bottom of the gradient. When flagellin
preparations are similarly examined, the protein is found hardly
to penetrate the top sucrose layer. Examination Of freshly pre-
:pared Solutions of flagellin in an analytical rotOr in the
ultracentrifuge show the presence of one slowly sedimenting
peak .of protein. The protein probably.exists as dimer with a
molecular weight about 301.000, so that an average size partidle
?of flagella might contain about 300 such units. Flagellin elutes
? as a single peak when chromatographed on a column of hydroxyl
apatite.
Amino acid analysis of flagellin from S.adelaide shown a
pattern of amino acids content similar to that for other strains
/4/. 'No cysteine is pre.sent? and there are 3 residues of tyrosine
per 215 residues of amino_ acids. The N-terminal amino acid is,
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Ada et al'. 4
alanine 'and the amount found suggests that the protein consists
of a,single polypeptide chain of moleoular weight about 28,000. .
The major chemical difference,b5tween the antigens is the Presence
: in flagella of the acid-insoluble moiety. This is riph in phospho-
rus And carbohydrate. A portion: of the fraction, containing some
? phosphorus, is soluble in lipid solvents. These properties are
-summarizedc,in Table 1.
? Serological _properties. Hyperimmune sera to each of the
three antigens were prepared in rabbits. The resulting sera had
' high titres as tested in the.bacteriaf-immobilization test of
NosSal /5/. Purthermore; each antigen in this test was capable
of neutralizing antiflagellar antibody prepared against any of
the three antigens. When expressed on a 'weight basis, flagellin
was Slightly less efficient as an inhibitor of anti.flagellar
antibody than flagella or polymerized flagellin, del diffusion
showed ane main common antigen and a second antigen which was
present in small amounts,.
:Experiments to test immunogenicity
Three general types of experiments were carried out. Two
'of them involved the injection of antigen into the hind footpads
-Of mature rats istar, albino rats, weight 150-250 g/. The thir,d.
? type .of experiment.involved,intraperitoneal injection of antigen
to neonatal rats and at various intervals thereefter. When mature,
those animals were challenged with antigen injected via the hind
footpAds. The amount of antigen injected varied over a 109
fold range, from 10'pipogram to 10 mg. /1 mg = 10-3g;
ljug = 10-,6g; l'nanogram /ng/ = 10-9g; 1: pieogram /pg/ = 0-12s./.
A minimum Of throe rats was used for each timq point.
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Ada et al. -5
'Rats Were bled from the tail at appropriate intervals up
to 36 weeks after the first injection. Serum samples were titratecl
for antiflagellar /anti-H/ antibody using the immobilization
technique desCribed previously. Initial dilutions were usually
1/5 /occasionally .1/2/. Pre-bleeds from rats were tested and
all showed absence of circulating antibody /titre < 1/5/.
Sera were tested for their susceptibility to the action of
Mercaptoethanol /0.1M0.37?C, 1 hour/. In sep\arate experiments
. /6/, it was demonstrated that mercaptoethanol sensitive antibody
sedimented in a sucrose gradient at ,a rate close to that estima-
ted for a 19 D protein, whereas mercaptoethanol-insensitive
antibody had a sedimentation coefficient of 7 S. Thus, for
the purposes of?this article, gross sensitivity to mercapto,
ethanol is regarded as indicating 19 S antibody.
? Antibody response to a primary injection /6/
"flagella. For doses in an intermediate range, the characteris-
tic finding was 'a lag period'of 3 days folloNed by a sharp rise
in antibaiy titre over a3-'4 day period. An apparent short *.
lag period was followed by a long slow rise in 'titre which.
at
reached a peak about 6 weeks. Thereafter, antibody levels stayed
high or decreased only slightly forperiods up to 34 weeks after
the injection. With low doses, for example, 1-10ng, little or
no antibody was detected in: the first two weeks and only mode--.
. rate titres were found thereafter. Antibody was not detected
in rats receiving 10 pg of antigen but some /6/45/ formed low
..levels following injection of 100 pg. The lowest uniformly immune-
-genic dose was'100,ng. Doses higher than this merely increased
the rate at which maximum titres-were obtained.
Most antibod5;. formed in the first week was macroglobulin.
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T
Antibody present at three weeks and thereafter was 7 S. Whereas
'there was little evidence of a dose response relationship over the
.range 1.00 ng to 1 mg for 7 S antibody production, there is a
dose response relationship for 4.9 S antibody production in the
'oange of 10 ng to 10 pg antigen. Figure 1 illustrates some.
of these results.
b/ Polymerized flagellin. The general features of the anti-
body response to polymerized flagellin were similar to those
demribed above for flagella and are not recorded here in detail.
Early production of macroglobulin was followed by-high and prolon.
ged titres of 7- S antibody. The major difference was that with
lower antigen doses,-about ten times as much polymerized flagellin
was needed to achieve the response resulting from a given dose of,
flagella. Thus, l/ug of the polynter, compared with 100 ng of
flagella, was the smallest uniformly immunogenic dose and the
smallest dose causing significant 19 S antibody production.
c/ Flagellin. The response to injection of flagellin, studied
over, the range 10 pg to 10 mg: differed significantly from the
response to the previous two particulate antigens. Figure 2 shows
the antibody titres obtained following injection of doses between
10 ng and 10 7ug, Points to be stressed are: there was no dose.
response relationship over this range. The lowest uriiformly, immuno-
genic dose was 10 ng, but 4 out of 5 rats injected with 1 ng also
responded. 'Peak titres were not as high as those given by the
, particulate antigen. After injection of 100 /ug or less, antibody
? was first detected more than a week later and it was mercapto-
, ethanol resistant. Injection of flagellin in doses higher than
0
this caused the early production of sma4 amounts of macroglobulin,
The necessity to use high concentrations of antigen to chieve this
. response suggested that it was due to a contaminating material,
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Ada et al. 7
posSibly polymerized flagellin..The evidence appears to
lnAicate that soluble flagellin, per se does-not cause the
production of macroglobulin. ?
? In this Connection it is worth noting that injection of
each antigen caused.the formation of Some anti-0 antibOdy and .
...this was macroglobulin. i.tres reached a peak in one week and
then slowly declined. As no dose-response relationship was
found, it is difficult to compare each antigen in this respect.
'Antibody response to a second injection
The general procedure followed was to inject mature rats
with antigen via the hind footpad, and nix weeks laterto
_give a'second injection Of antigen by the same route. Blood
samples for antibody estimation were taken at intervals, parti-
cularly immediately prior to and after the second injection.
Using suitable controls, a rapid response and increased titres
were taken as avidence of a secondary response. It was hoped
.to answer the following types Of question. In view of the pro,i.
:longed response to a primary injection of these antigens, can
a second injection give enhanced antibody titres and if so,
what
-
-what .are the relative amounts of antigen needed te achieve this?
Can an enhanced response be achieved if the primary injection
is a ?"sub-immunogenic" dose?- What type of antibody is formed
in a secondary response? Is flagella more efficient than hagel-
lin in causing an enhanced response? Can flagellin enhance
A response initiated by flagella? Not all these questions can
as yet be answered in detail'. Attention so far has been con-
centrated on experiments using flagella and to a lesser extent,
' The results using two doses of flagella fall into two
?
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Ada et al. - 8
'
' . groups. In h
those animals which receitted an initial'doSe of
,100 ng or more.of-flagella,:the serum titres at the end of slit
weeks-were already substantial. /about 1000,4 and were duo to
7 5 antibody. dhallenge,with adose"of flagella Smaller than
this'resulted.in,titresvhich,were in the same range as.-the,
.control values Higher titres'were Obtained onlTif the challen-
. ge dose was equal to a greater than the dose of .flagella previous-
v
17- injected. This is shown in two experiments quoted in Table 2
/lines 4 and, .Becaise of .the high initial. .7. S antibody titres,
we have not been able to determine how much, if any, of the new
. antibody formed wasinacoglobulin, The situation was more clear
,ctat when the initial dose of ,flagella resulted in either trace
' amounts or no antibody titres being detected at the aix week
period.. Table 2 /lines 1-3/ shows the results of two such
experiments. The initial dose was 1 ng in each case and the,
second dose, ?el.ther 1.ng or 10e/ug; /n both experiments,- there
. -
?
was an immediate marked response to the second injection. Further-
more,. all the antibodytprodUbed?:up to 5 7 days after the. second ?
injection was macroglobulin,.Ratagiven ].'or - 10 pg an initial
dose of flagella (11..a.nohcm Tiny enhaneda'response upon challen-
.
''ge.jiesults.obta.inedusing'100-pgof flagella as the Primary
? Mose, have been very variable.--,_d one experiment., challenge
'with 10 ./ug of flAgell'a or pOlyffierized flagellin gave rapid,
?
enhanced.responses,and the antiOdy fon-fled was mercaptoethanol-
senSitive. Other experiments ? of.a.similar nature did not. show
such an enhanced response.
.Eperiments .using
?
far advanced as those
,reported above.. It ip clearthatrats 'tivbn, a-prior injection
.'2H---- ?
of flagellin /10. Ald:will,show a--VerY''1/16rked reaponse when
I
challenged with a similar des.eOf lIdg6iiiri polymerized flagel-
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Ada et al, . 9
lin or flagella /Table 'W. Experiments using small doses of
flagellin in the .initial injection followed by varying doses
Of flagella .or.flagellin are, in progress.
[
lin, polymerized flagellin or flagella. After 14 to 16 weeks of such
treatment, no antigen was given for 8 weeks and the rats were then
challenged with a signle dose of the' same antigen. Sera were taken
'throughout this whole period and afterwards for.the estimatiop.of
antibody.
The results show beyond doubt that rats can be made tof:qrant
Nto flagellin and at this dose level remain in a tolerant 'state
Acquired tolerance to bacterial protein antigen. Th d condition
of tolerance is recognized as a Continued failure of the host ani-
mal to produce. detectable' antibody to injected antigen. The classi-
cal procedure for producing such tolerance is to inject in 'pingle
1T7
or multiple doses substantial amounts Tof antigen into the Vnal
either prior to or shortly after birth. Tolerance to many anti-
gens has been achieved, but no one has yet reported complete tole--
ranee to well defined proteins of bacterial origin. We have attempt-
,ed. to achieve tolerance to the antigens described above. In most
experiments to date, the procedure has been to inject rats on
the day of birth and at frequent intervals thereafter. After some
weeks of such a course, the animals have been rested and then
challenged by one or the other antigen. Control experiments in..,
volved challenge of normal 100 week old mice with comparable amounts
of antigen. Table 4.showis the results of experiments in which rats
were given at birth and then twice a week, ?.0 ,ug doses of flagelv
for some weeks afterantigen injection has stopped, Some remain
tolerant even after a further dose of'antigen,is given Rats given
polymerized flagellin develop gooa partial tolerance which stands
up well to subsequent challenge. On the other hand, rats given
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Ada 'et al. - 10
flagella from birth rapidly develop high antibody titres. Despite
this, rats receiving-'10/ug of flagellin twice a week, when
challenged with 100 /ug flagella at 11 weeks, produce a small
amount /titre 10.to 160/ of-macroglobulin Only and this dis-
appears rapidly from the circulation.
How much antigen is necessary to confer a tolerant state
in rate Rats Were treated in the same way as above but the dose,
of .21agel1in varied from 100 ng to. 1 mg twice a,week. Injection
of 100 ng quantities led to high antibody titres in 6 out of 7
rats. Good tolerance was achieved with the other dose levels. At
the higher dose levels, particularly 1 mg, there was early pro-
duction of small amounts of antibody and 'this was 19 S. Rats
treated at the 100 /ug level until 16 weeks old remained almost
completely tolerant, even when challenged at 24 and 32 weeks
with 10 lug doses of flagella. Similar experiments with poly-
- /
merized flagellin are under way and hgve already shown that
N'
at the lower dose levels /1 ng, 100 ng/, injection of this antigen
leads to high antibody production.
- Finally, a further development is the determination of the
amount of antigen which will induce tolerance to' flagellin in the
rat given as a single injection on the day of birth. Such informat-
'ion will allow a meaningful .study to' be made of the distribution
and fate of the antigen' in these circumstances.
' Discussion
The experiments reported in. this paper are.part of. a programme
to study the induction of antibody formation bY -small amounts of
powerful antigens. Bacterial antigens were chosen for this reason
and also because a study of their behaviour has special relevance
to' practical immunology. Three related bacterial antigens were
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? Ada et al. . 11
prepared and characterize# flagellin, a soluble protein of low
molecular weight; polymerized flagellin, a linear polymer of
this ,protein; and flagella; the naturally occurring particle
from which the other two antigens were derived. Though each
..preparation.caontained the;saMe protein unit, there were major
differences in their size and structural organization. As an
initial step in the overall programme, the effect of these
: variable on the ability of'each antigen to cause antibody format:-
ion after a primary and secondary injection and to induce immuno-
logical tolerance, has been investigated.
A first major finding was that with each antigen, a single
' injection of a,remarkably Small amount caused the production
of detec,table antibody. As these proteins are. bactei,ialprcducts,
. an immunological reSponse to such small amounts raised the ques't-
? ion whether in-fact we were studyinga primary response or
a secondary, enhanced response following a primary natural in-
fection. Since specific antibody has not been detected in a
-pre-injection bleed of. any rat, three pieces of evidence present-
ed in the paper support the primary response hypothesis. 1:
Injeotion of antigen results in very prolonged antibody product-
2. Injection of doses of flagella many times, less than the
? Immunogenic does does,not result in immunological memory.
3: Tolerance has not been achieved with any dose of intact_
0
flagella or with micro-doses of flagella derivatives so that
a natural, early infection Should have yielded detectable anti-
r'-body titres. Twe other points are worthy of special comment.
. The particulate antigens produced early 19 S antibody and
subsequently, high titres of 7 S antibody. In view of the chemical
'similarity between flagellin and polymerized flagellin, size of
the antigen appears to be a major factor in this connection'.
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Ada et al. - 12
.The second :point is that though the particulate antigens produced
higher-7 .8 titres than aid flagellin; lower doses of the latter
were more consiltently immunogenic. Thib was particularly so when
flagellin was compared with polymerized flagellin where the dif-
ference was about 100-fold. That is, in this respect one molecule
.of flagellin seemed to' be equivalent b one particle of the poly.
mer, I.is known from. autoradioagraphic-studies17, 8/ that
flageilin diffuses more readily through the popliteal node than
do the other preparations so a low moleculare size may be of
particular importance in this respect.
The studies reported on the secondary response to these
proteins are. incomplete so, discussion will be'confined,to those
0
obtained, using flagella as antigen. Two points are of particular
interest. Current results indicate that an enhanced secondary
response was obtained only if the -challenge dose was. equal to
or greater-in amount than that given in the first injection.
This is turn suggests that to elicit such.a response, the level
,of antigen. is the.lymph nodes must be substantially increased. It,
is. known from studies with i131 labelled flagella that six weeks
after the first injection, the level of antigen in the popliteal
node is about. 25% of that present in the node 24 hours after
injection /9/. This may mean-that an increase of about 10 fold
2
in the, amount of antigen in the node has given an enhanced response.
%
The second point is the clear demonstration of immunological memory
.for 19'S antibody when flagella was used as antigen. This was
-shown when tho initial injecion of antigen was a dose near Cr
slightly below the minimum immunogenic dose so that. either no
or very low levels of antibody was detected at the six week period.
Bauer,-Mathies and Stavitsky /10/ did not observe this effect in
studies with Salmonella nor did Uhr and Finkelstein /11/ using
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Ada et al. - 13
bacterial virus as antigen Uhr and Finkelstein suggested antigen
.deiiletion.;as a:poSsible reason.for the failure of the cell respon-
sible for 19 S antibody formation -to develop persisting immuno-
logical memory, but until information becomes available on reten-
tion_in_lymphoi_d_organs of the responsible- antigen in the bacterial
. :
...virUsi.'thotwo'systems cannot be .compared in this respect.
,The achievement of tolerance in.rats to a purified, highly
imMUnOgenic bacterial protein op.enthe..w. y for meaningful in7
Testigation.into the.mechanisms of tolerance, the fate of the
protein. intolerant and nothrial animals and in respect to the
present thesis, the antigenic complexity of the reagents. The
results presented are incomplete but several observations can be
Made.'dhough complete tolerance to flagellin has been achieved
'in rats for extended .periods, injection of-polymeriZed flagellin
? over a range of doses has yielded good but not complete tolerance.
_Agaln:the effect of size of the antigen is demonstrated..Can
flagellin, because of its small size, gain access more readily
to all the ?critical sites in the body?. Both preparations when,
given in frequent large doses after birth csue the formation
'of small amounts of macroglobulin. An explanation Tor this may
be the. presence in both-samples of trace amounts of a substance/s/
present in greater amounts in the flagella. The failure to obtain
T,
tolerance to flagella is of great interest. First, it seems not
unlikely that this is of considerable biological significance
with:respect to the maintenance of the animal's integrity. Second-
ly the finding that animals tolerant to flagellin are essentiallY'
.tolerant to flagella - a-small amount only of 19 S antibody is
formed indicates clearly the similarity of the main antigenic
grouping in each preparation. Perhaps the failure to achieve
tolerance to intact flagella is due to an adjuvant action of
S. \
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Ada et al. - 14
substances in the acid-soluble component. Further studies-may
show this,
In conclusion, the results reported have shown clearly'
?ferent effects given by Soluble and particulate forms of An anti.
gen. Many of the questions asked however remain unanswered.'.0nel
which is at the basis of all this work, and is particularly
intrigUirfis why is an apparently simple protein, such as fla-
gellin, such a powerful immunogen?-
.'.SUmmary
1. The preparation andlDroperties of three related bacterial
antigens has 'peon described. Flagella, .obtained from Salmonella
adelaide were brokeir'dOwn to form the soluble protein,. flagellin,
which inturn was readily aggregated to form .polymerized flagellin.
,Flagellin and polymerized flagellin were chemically siMilar but
flagella contained in addition a substance rich in carbohydrate
and phosphorus.
2. Each preparation was a powerful immunogen in rats.
.The particulate a4igens, flagella and polymerized flagellin, caused
After one injection early 19 S AntibodY formation and then high
and prolonged titres of 7 S antibody.. The soluble *antigen,
flagellin, Caused only 7 S antibody formation and lower titres
than those caused by the other preparation. In contrast, a smaller,
dose of flagellin than of flagella or the polymer caused con- .
sistently an immundlogical response..
3. A second injection of flagella or flagellin caused an
enhanced response. Persistent immunological memory td 19 S antibody
formation was demonstrated when the primary injection of flagella', '
was a.dose near or. slightly below the minimum immunological dose.
4. Complete tolerance could be obtained to flagellin and good.
FCBEFFICM -UFO Y
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Ada et al.
parti41 tolerance to ,polYmerized flagellin., In constrast, a '
1-simi1ar course of injeetion !of. .flagella caused high antibody
titres in the recipient rats; it was -cif particuiar interest
however, that rats made tolerant to flagellin were- also
. essentially tolerant to flagella.
.References
? /
1. Astbury, W.T., Beighton, E., and Weibull, C. Symposia.
Soc.Exp.Biol No.9 :282 /1955/.
Ada, G.L., Nossal, Pye, J.,? and Abbot, A.: -AuSt.
J. exp .Biol. /in press/.
...3. Kobayashi, T., Rinker, -J .W? and Koffler, H.: Arch.Biochern..
Biophys. 84: 342 ./1959/.
4. Ambler, R.P. and Rees, ,M.W. : Nature 184: 56 /1959/.
.74 Immunol. 2:137/1959/.
5
. 6. Nossal,? G.J.V., Ada,- -G.L., and Austin, C.M. :Aust.J.exp.
Biol. /in press/ '
7. .Nossal, G.J.V., Ada, J.,. and Austin, C.M.:Aust.J.exp.
Biol. /in press/.
Ada, G.L. , Nossal ? G.J.V? and Austin, C.M. Aust.J.exp.
Bidl. /in press/. ?
. . -Ada G.L. Nossal, and: ?Pyel
/in press/.
lb. Bauer, D.C., -Mathies, Mp,T;in and Stavitsky, A.B. :
Med. 117:889 /19,63/.
*exp.
11. Mir, J.W. and Finkelstein J.exp.Med.11 7:457 /1963/i
? , ?
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Ada e
Properties of three related bacterial antigens.
Atigen
Protein Composition
oCarbohydratesPhosphoru
Flagellin Soluble, Soluble, low
0.2
0.0025
? ?
Polymerized
flagellin
Particulate, rods of
varying length
< 0.2
< 0. 0025
Flagella
Particulate, rods of
varying length
1.1
0.03
,
-
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Table 2
4
Ada et. al, - 17
Antibody titres of the. sera Of mature rata given two ?d0Ses,
of flagella,
?
Each 4.1tie the geometric mean of three results.
?
Primary
?
Dose Antibody titre
-
injoctedi. at six weeks -
/prior to second:
injection/
_
Secondary
Dose .
injected
4
1 ng.
1 ng.
NIL
100 ng
10 /ug
5
-not detected
not detected,
800
2,400,
.
-
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ng
10 /ug
10 /ug
10 Jug
,..(
10
/ug
\ '
-
, Antibody titre,
after injection
4 days 5 days 7 days
' . 25
400
40
2, 5 60
16,000
1,280
'100
,140
6,400 _
32,000
SIMI -
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,
?
Ada.et al. - 18
or.
H
Antibody.titres? of the sera of mature .rats given.an, injection of
flagellin, and six weeks later given an injection .of flagellin,
?
polymerized flagellin or flagella.
?
Each value is the geometric mean of these results.
Primary
Dose Antibody titre .
- inlecto d at aix -weeks
80
Secondary
_
Dose injected
? Antibody titre
after injection
4. days 5 days 7 days
.10
,10
,ug flagellin
1,280
7,680
10 /lig polyme-,
rizbd flagellin
41000.-
20,000
,ug flagella
2,400
5,120
11111 tt, P.tk
.-1Ava
-
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. - r? ? . .
Table 4
?? ?
e
tr?
?-?
? t.
Imm4no1ogical response of ;rats injected since birth with flagella,
loolymerized flagellin' or flagella.
.Ratb were given twice weelPy doses of antigen for 14-16 weeks - ?
Ada et al. 19
Period. 1. ?
.N0 'antigen was'then given for a periodot. 8 weeks,- Period 2.
'One further injection /10
7
, given and antibody' titres 'estiMated- 2-4 weeks;later - Period 3. ,
Titres olioted are the geometric mean of values from 3 or more
rats - the range of titres' is given in bracketS.
tag/ of.the?same antigen was then
Antigen pose
AntibodY titres
;Peribd 1, Period ,2 Period
10 weeks ? 14 weeks 16- weeks
Flagellin 10)bg kvai4i1.0 F.: Folia Microbiol.
8: 197 /1963/.
.Eisen, H.N., Kern, M., Newton, W.T.,and kelmreich, E.:
J.Exp.Med. 110: 187/1959/.
9. Farah, F,S.,, Kern M and Eis.on, N.: J.Exp.Med. 112:
1195 /1960/.
Benacerraf,
J.Exp.Med.
k.,
B., Ovary, R., Bloch, K.J., and Franklin, .C.:
117: 951 /1963/.
rank, 'F. and Lanka,6 V.I Collection Czechoslov.Chem.Communs.,
28: 24.5 /1963/.
12.' Frank, F. and J.ictin,
29: 1401 /1964/.
J.: Collection Czechoslov,Chem.Communs.
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. . ?
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..?
?
Frank et al. - 15
13. Eclolman, ?GIL, and Poulikr,M.D.: J.Exp.Med?,113 861
/19 61/,..
14. :Cohen, and liorter,,:,R,R.: Bioehem, 4 901 278 /1964/.
/ ?
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7.
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? ru4 urriumL uniu,
? Frank et al. - 16
Legendes to'-figures
Fig.l.. Starch gel'electrophoresis of bovine and pig antibody
.? ? ,
andiamma-globulind and their.derivatives.-
Composition of buffer: 0.05, M. formic acid,- 6 M.urea. 1 -; bovine
-gamma globulin, 2 .S.!_suiphO. bovine anti-DNP antibody,
S-sulpho bovinc. gamma glebulin,.4 S-sulPi).o pig.gamma globulin,
5.- S7sulpho pig ant-DNP antibpdies, .6 - pig gamma-globulin.
Starch gel electrophoresis of bovine t.003.-g1obt4in and
anti-DNP antibodies- S-sulphonated-at pH 8,6,
:Composition of buffer; .0,05 M formic acid, M urea, 1 - gamma-
antibody, antibody S-sulphonatedin.excess
'dinitrophenol,. 4 - antibody,S-sulphonated in oxdess E-DNP-,
lysing.
I
CFig.3. Starch gel electrophoresis of bovine gamma-31ebu1in and
. anti-DNP antibodies .S-sulphonated-at pH 5,7.
,Composition of buffer:, 0.05 M forpic acid,. a, M urea. 1 - gamma-
globulin, 2 -.antibody 3-antibody S-sulphonated-in excess
dinitrephenoll 4 - antibody Z-;sulPhpnatTd in excess E-DNP-
lysine.
Starch gel electroPhoresis of L chains of gamma-globulin
. and antibody. Composition Of buffer: 0.036 M glycine buffer
? pH 8.8, '8 M urea 1- chains of nonspecific bovine gamma
:globulin,. 2; 3, 4 - chains of isolated.anti-DNP antibody from
three individual bulls.
-
T1g.5.- .Chromatograph 571 of S-sulpho bovine gamma globulin and anti-
DNP.antibody on Sephadex 0-100.
Medium: 0.05M. formic acid, .M urea, Abscissa: volume of elate
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?
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- A
!
Ordinate: optical; deneity at 2537 ./registered by Uvicord LX13/.
, ? .
Crosshatched area denotes fractions pooled, Different sub-
units denoted by letters.
Activity Of subunit S of bovine anti-DNP antibody
'S-s4phoWed at different
Medium for separating subUn4s:. 0,65 ,M formic acid, 6 urea.
Abscissa: volume of eluate. Ordinate left: optical density..
at '2537 R. Ordinate .right, value of T. Upper part Pre-'
'
paration Osulphonated a] pH. 8.6, lower 'part Preparation
Frank et al. -! 17,
8-sulphonated at pH 5.7. Activity expressed by.quantities
''of:r are depicted as ,crosshatched oll'Ithns and given in
-numerical values aPpendedi to the separate subunits.,
Fig..?. ? Activity of different 'mixture s dT H and L subunits of
bovine anti-DNP antibody. Abscissa: percentage of subunit
in mixture. Ordinate: activity. expressed as qu.,,ntity r.
\
HUpper part- hypothetical Curves, corresponding to 'ideal case
, .! ? , ?
04 very firmly bound H and I chains in stolchiometric
complex. Lower part . experimental cure,
_
Polarographic registration .of reaction Of.-DNP-.1ysine
with Complex of H, and:L chains of bovine antiTimp antibody
Abscissa: time interval' from mixing of protein with hapten.
L.,:Ordinate: concentration .of free E-DNP-lysine. Total amount '
of protein in experiment 2.5 mg. 1- .hapten 'added immediately. ?
af.ter mixing H and L chains 2 hapten added 20 lours aftar
\
mixing H and L chains.,
. , 5. ? ? -' " _
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?,.-/ ?
1.!
Frank et al. - 18
? Table 1;
.tc..c.b,ivity of 'bovine'. antiODNP antibodies and their derivatives
N. . Method of r Relative activity
modification
1 Native
"
2.
S-sulphonated
86,. 20 hour./
.a/ clissoled in: \?
borate buffer
b/ dissolved in 0.05 M
formic. acid with 6 M
urea and transferred .
after 20 hrsi, in borate
buffer
.1.77
1.40--
10..86
100
?
,. 49
+After reaching equilibrium with 1. X 10" M
!
.1
?
1.I.
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"s1
^
Table 2
F.'ranok 'et al, 19
.Activity of mixtures of bovine, anti-121U antibody and bovine
gamma-globulin chains
.1
TyPe'Concentration
, .
.of + in mixture
chain mg/nil
Total
concentration
of protein
mg/mi
r++
rH+++,?
? -F.++
L '
Hsp 0.50
sp 0.50
H 0.33
sp,
+ Lai 0:17
H +' 0.33
.+Lg ?0.17
Hsio+0.3
0.,85
Hg +' 0.33
+Lsp 0.17
,.
Hg 1.00
+L ? .0.17Y
sp
0.50
0,50
s
0.50,
0.50,
. 148
0.50
1.17
0.00
0.06
0.58
0.34
D,036
0.11
0.06'
'0.00
0.58
0.34
0.86
1,
Q.09
0,58
0.11
0.1:4
+ Hsp Lsp chains of antibody Lg - chains of nbnsp.ecific
,
-,gAmmaglobulin
++.0orrpcted.gor non-7spocific adsorption,
rH, and rL represent moles ofrDNP.:.lysine bouhd to
107,000 g of.Hsp chain'and to 53,000 g of Lsp chain
respectively.
'
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The. Ailotypie Determinant's of Rabbit- Gamma-GlobUlin,
tS Fragments and Antibodies
P4GH.Gell and A.SiKelus
The UniVersiti. of Birmingham, ? Birmingham Endland
Abstract.
' The allotypic determinants /AS/ of rabbit Th.-globulin are
controlled by. two gene lo6i a /As 1, 2 and 3/ and b -/As 4, 5 and.
6/.
1 1. Work already published /1/ has indicated that i/ the
Light. /B/ chains' appear to: contain only the determinants control-
led,by the. b locus, ii/ the Heavy /A/ chains appear; within the
limit-tions of the technical methods available, to contain, in
most casos, both the :determinants controlled by thea and those
controlled by the t locus. As 5 fd;ed a partial exception to' this
in that we could only demonstrate it in. the A chain, from one hemo-
zy6ous /As 3/5/ animal; and failed to find it in four animals .-
? -heterozygous at this locus, although As 4 was demonstrable in
thee preparations. Similarly in .one preparation from as
As 1/4/6 animal., AS 6:-was demonstrable but not As 4. .
We 'have also' recently ./2/ been able to confirm by gol!-. dif-
.fusion Todd's /3/ observations, made using the interface /ring/
test; that in rabbit =',-macroglobulins, As. I may .bo present as well
as the determinants /AS 4, 5 or 6/ present in the B chain f we
were able to demonstrate AS 2 and As 3 in Addition. ThcSo macro-
globulin preparations were made by a combination of SG 200 gel
-
filtration and starCh- ;block' electrophoresis: they reaCted with
. an antiserum containing antibody against '-macroglobulin, but
with ono against S' 7 j-globulin exclusively.
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Gell et al. - 2
Theoretically one might forecast that the allotypes control-
led by the a locUs would occur on the A chain only and therefore
?
not in macroglobulins, which possess a.distinct kind of A chain:
and that those controlled by the b locus would appear on the
. ?
B chain only, and therefore should not be demonstrable in A chain
Preparations, That neither. of these forecasts is fulfilled May
reflect either Upon the techniques currently available for fract-
ionation of proteins and their fragments, which may still be in-
adequate to effect really complete separations, or alternatively
upon the oversimplicity of the genetic theory upon which the
\.$
forecast was based*
2. Antisera to allotypes may be raised /4/ by,immunizing
a donor rabbit with bacteria, coating a 9uspensl,on of these
bacteria with the antiserum so raised, and injecting the- ,-
globulin /antibody/ 7 coated cells into a'recipient rabbit.
In an attempt to identify new 6-.11otypeS, we carried 'out this
"4.
process using As 1/4 rabbits only : and have recently published.
/5/ some preliminary results of this.study, which are essential-
17 similar to those a? Oudin and Michel /6/.
Antibodies were indeed proauced in four out of five As 1/4
'-rabbits immunized from an As 1/4. donor /D/, itself immunized.with
'Proteus vulgaris, but these antibodies were found to.react,only.
with the immune /Dl/ serum, not with a pre-immunization sample
from D, nor with the blood of about 80 relatives'or progeny. of
?
D. The positive reactions cannot therefore be due to the presence
of a new allotyPe determin-ant_occuring in D but not i R, but
must be contigent upon the immunization of ra? bbit D. The substance
present in Dl and antigenic.in ? :R1 could be shown to have the
physicochemical characteristics of a' q 7 ''r-globulin : it would
FOR-CFRira; ? ".117;
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- ? A.
r`ft
' Gel et al - 3
:teact asantibody with ProteUs vulgaris extracts and as antigen
ke),..-:globulin/ with antiAs.1 antisera I moreover by gel diffusion
tests it. appeared that all the Rl-reacting substance is anti-
???
'Proteus antibody and all the anti-Proteus antibody wab Capable
of reacting with Rl. Full absorbtion of R1 with Proteus *extract
or cells did not affect ,the Rl/D1 reaction, which was however
abolished by absorbtion of D1 with proteus extract.
By immunoelectrophoresis and agar block electrophoresis
followed by extraction, the reacting DI substance was found to
have a sharply restricted mobility within ,the globulin range
and in this respect resembled a myeloma protein.
R1 wa4.not an anti../anti-Proteus/-antibody in the strict
sense, since it did not react with, several strong anti-Proteus
antisera and indeed had itself appreciable anti-Proteus activity'.
,These and other data les us to postulate that the Dl substan-
ce was a 'clone product! selected by immunization and present if
at all only at undetectible concentration in the original pre-
immunization sample. Further experiments -along these Imes are
? being made and will be reported.
Reference
1. Fleinsteinv A., Gell, P.G.H., and Kelus, Nature 200:
653 /1963/.
2. Feinstein,--A.,'?aell, P.G.H., and Kelus, A.S.,: to be published.
3. .5041.0 C.W.: Biochem.biophys.Res.Commun. 11:170 /1963/.
.4. .Dubiski, Ss, et al..: Immunology 2: 84 /1959/.
Do Gen,. and Kelus, A.S.: Nature i687 /1964/.
6. Oudin, J. and Michel, M.: C.R.Acad.Sci, 257: 803 /1963/.
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Heterogeneity of AntibodUs in Alergio Sera+
A.H.Sellon
Department of Chemistry, McGill University, Montreal, Canada'
Introduction .
As in acquired immunity, the charaoteristic feature of
allergic reactions in man, of both the immediate and delayed
type, is their spedificity with respect to the particular
allergen. Since the introduction of the passive transfer test of
Prausnitz;and K?stner in 1921/1/, it has been generally accepted
that hypersensitivity states of the immediate type./suoh as
hay, fever?,asthmay.food allergiQs/ are associated with, the
prespnce of humoral skin-sensitizing antibodies, produced
.UontaneousW by the allergic individual in response to the
.caSual exposure of a given allergen by either inhalation or
ingestion. Moreover, since 1935 /2/, it has been recognized
that treatment of 'allergic individuals4trwith a series of
injections of the offending allergen leads to the formation of
+The work done 1n the author's laboratory and referred to in this
paper has been supported by grants from the National Institute of
Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, 1Md., and the Medical ResearCh Council of Canada and
the National Research Council of Canada Ottawa, Ontario.
++Allergic individuals who have received at least one series of
injections of the allergen are denoted as treated individuals;
prior to such treatment the allergic patients will be referred
to as non-treated-individuals. For the sake of brevity, sera of
allergic individuals will be alSo designated as allergic sera.
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Sehon - 2
of an additional-humoral antibody., termed blocking antibody,,
which was shown to be capable of neutralizing the allergen.
In addition, to skin-sensitizing and blocking antibodies, which
are demonstrated by the in vivo P-K test and by the blocking
of. this test, iiespectivelyl.the'presence of agglutinating anti-
bodies has been regularly demonstrated within the last decade
in the sera of both non-treated and treated allergic indivi-
duals. by passive hemagglutination methods /304/.
By contrast, so far, delayed hypersensitivity has not
been passively transferred with serum, but only with the -son-
?
,sitized donor's lYmphoid cells or, ih the case of tuberculin
sensitivity in man, with the corresponding cellsextracts /5/.
-Conpequently, it has been postulated that delayed hypersensitivi
ty is mediated by a cellular antibody-like factor/s/, referred
to as the transfer factor/s/.-The presence of hemagglutinating
antibodies had been demonstrated along time ago /6/ in sera
-of individuals with tuberculih.sensitivity to.PPD /Purified
protein derivative of tubercle bacillus/0 and recently it has
been shown that these sera contain at least two physico-chemic ally
:distinct hemagglutinins /7/.
The distinguishing features of skinsensitizing and block-
ing reviewed at the First Symposium hold in
.Prague five years ago /8/ and were summarized in a table, which
is reproduced here /see Table 1/, since the data listed in this
table have been' confirmedby many workers and since it represents
a convenient starting point for thee present discussion., This
paper r1,11 be, therefore, confined primarily' to a presentation
-of additional physico-chemical, and immunochemical properties."
of skin-sensitizing al-id. blocking antibodies, which have been
unravelled in recent yours, and' to an analysis of the possible
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;
Sehon - 3
relationships between ,those antibodies and the hemagglutinins
invariably found in the sera of allergic individuals. In spite
_of the great variety.of-allergens, most of, the systematic studies
of the properties of allergic antibodies have been done with
-sera of grass- /9, 10, 11/ and ragweed-sensitive /8, 11, 12, 13/
Individuals. As will become apparent, the exact nature of these.
antibodies and the mechanism of their in vivo and in vitro inter-
actions with the homologous allergens remains still undetermined,
partly because of the-eXtromely .small concentration of these
-antibodies in allergic serao'partly because of :the failure to
r? ?
"isolate them in a pure state, and partly because of the un-
availability of pure allergens. Nevertheless, it has become
evident that-1, the elder differentiation of .sera of non-
treated allergic individuals 'from sera of treated patients is
purely arbitrary and outmoded, 2. both types of sera contain
.mUltiple /and probably the same type Of/ antibodies, and 3.
these bora differ mainly in the relative amounts of the
various antibodies rather than in the quality of the antibodies
produced /14/.
I
Skin-sensitizing antibodies /REAGIN/
Physicechemical.behaviour
. As was previously established. /15/, all observations to
date indicate that skin.sensitizing antibodies are much more
labile than "conventional" antibodies produced on immunizat-
? ion. Even, seemingly harmless. procedures, such as dilution- of
sera and reconcentratio# by. ultrafiltratioff, .orb/ pervaporation
-Combined with dialysis, leads to serious losses or reaginic
.activity of the order of 30_50% /12, 16/. Like immune anti-
'bodies skin-sensitizing antibodies are degraded with papain..
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?
^7,
t .
t ?
Sohon,- 4
? However,, whilst immune antibpdies are split very rapidly,
.i':1._withinthe first 30 minutes of digestien 99% of those
? antibodies are destroyed /177; skin-sensitizing activity .remains
practically unchanged within the first.30-60 minutes of digest-
on and dperease gradually requiring a 24-hour\poriod of digest..
ion for complete inactivation of roaginic antibodies /17/,
linlike'immune antibodies, skin.sensitizing antibodies ?
are inactiVated in the absenee.of papain by treatment with
,.mercaptoethylamine at a concentration of 0.1M /17/. This in-
!
-activation appears to be. the result of the reductive cleavage
of skin-sensitizing an.ebodies since treatment of allergic
sera with lower concentrations of the reducing agentor with
iodoacetate alone /which is used for the stabilization of the
SH groups liberated'during.the reductive cleavage/ does not
lead to a measureable decrease of skin.sensitizing activity.
'..Xhe results obtained in the studies on the enzymatic and chemi,
cal degradation of skin-sensitizing antibodies indicate that
? during these reactions the portion of the skin-senizing ?
antibody molecule responsible for its attachment to the skin
becomes destroyed or 'dissociated from the rest of the molecule
/17/.
Serum fractions enriched in skin.sensitizing activity were
isolated by chromatography' of allergic serum on 1. the anion-
exchange resin diethylaminoethylcellulose./10EAE-cellulose/ .
/110, 12/ and 2., by gel filtration /18/ on the cr0887linked
dextran, Sephadex G-200. Thus, whilst blocking antibodies were
almost quantitatively recovered from DEAE-cellulose in the first
chromatographic fraction /eluted with 0.01M phosphate buffer at
pH 7.5/, skin-sensitizing antibodies were eluted later in fract-
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\s.
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Sehbn - 5
Ions with buffers of higher molarity /0-02 and 0.05M/ and of
lower pH, /pH. 6.2 and 4.5/. These results confirmed the. previous
findings /19/ that. skin-sensitizing antibodies were physico-
' Chemically heterogeneous and additional electrophoretic /10, 20/,
ultracentrifugal /B. 21/ and immunochemical /10, 18, 20/ analyses
.revealed that the fractions possessing .skin-sensitizing activity.
Were complex mixture of Proteins.
Fractionation of sera on Sephadex G-200 yielded three
fractions which were not resolved completely from one another
and which were composed of 1. macroglobalins /rim- and
ct2irg1obulins/, 2. other globulins in-. Ai- and 4(-globu-
lins/ of lower molecular weight, and. 3,albumin.? UsinOhis Method
-for the fractionation of sera of ragweed-allergic individuals
/18, 22/, skin-,sensitizing activity was found pritarily in the
early: eluates bf the second fraction, i.e. in the eluates correspc
ponding to the region of overlap betwetn the first and second
chromatographic fractions, the bulk of skin-sensitizing acti-
.vity emerging before the peak contaltiAntiohinCatha1t6o6ng
fradtion.. By immunochemical analysis, .he eluates 'containing
.skin-Sensitizing activity were found to be composed mainly
of r -globulins and 7 S r-globulins, and contained also
lA
,a small amount of /1D/1-globulins and possibly other components.
These results were interpreted /18/ to indicate that the
.distribution of skin-sensitizing activity parallelled best
that of 7,1A-
globulins, a conelusion which was in agreement
with the earlier suggestions, of Heremans /23/ and of Augustin
and Hayward /10/. .
The interpretation of the experimental data concerning the
ultracentrifugal properties of skin-sensitizing-antibodies has
led to much controversy. The reason for this controversy might
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f -
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f.,3a CFFICIAL 1/Z. E4-ar
9
FOR tVVV:41.;'11 USE ORLI
1
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4
0
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?
?
.Sehon- 6
be due to the fact that in these investigations sera of?
?
'individuals with different allergies /lye. to ragweed and
grass pollens, horse dandruff, rabbit and .cattle hair, molds,
glucagon/ were used and, in most of these studies, no syste-
matic discrimination was made as to whether the sera had been -
?
obtained from non-treated or treated allergic individuals; more-
over, usually no special mention was made of the length and
method of hyposensitization treatment administered. The dis-
crepancies in the observations may, therefore, merely reflect
:
the heterogeneity of skin-sensitizing antibodies produced to
different allergens and under different conditions of exposure.
Furthermore, the techniques.empnyed in practically all these
studies were different /i.e. fractionation of whole serum by
preparative ultracentrifugation, ultracentrifugation in a densi-
ty gradient, ultracentrifugation in partition 'cells .and ultra-
centrifugal analysis of serum fractions containing skin-sensit-
izing antibody/, rendering the corPlation?of the results dif-
ficult,
As was stated at the Fivst Symposium /8/, the analysis of
the distribution of antibody activities in serum fractions
isolated by ultracentrifugation in partition cells indicated
that reaginic activity Was associated with serum components.
having sedimentation coefficients largerthan :,.,7 8, the actual
values measured in terms of P-K titers being 12.4, 14.1 and
22.5 S /21/. However, because of the inherent inaccuracies of
the titration procedures used, all these values were considered
accurate only within 2:5 s /21/. These conclusions were challen-
ged by_a number of workers /10, 20/, all of whom favoured the
value of 7 S for the se-dimentation coefficient of reagins.
1. ?
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Sehon - 7
Sine() none of these conclusions was based on an unequivocal
evidence, the suggestion was made that skin-sensitizing activity
,was nbt associated with either 7 4 or 19 S serum components, but
with other moieties hacking sedimentation coefficient/s/ inter-
mediate between these Values /24/. Obviously, such components
-Would hive ,been detected either with a light or with a heavy
serum cemponent depending on, the experimental conditions used.-
. Indeed, recently, using an improved method of density gradient
illtracentrifugation /25/, skin-sensiti2ing antibodies 'to gluca-
.gon,were found to sediment with serum Components having.sedimen-
tation coetficients of the order of 8-11 S. Similar conclusions
'were reached also by Terr and Bentz /22/ from an ultracentrifugal
study with four sera of untreated ragweed sensitive patients and
an average sedimentation coefficient of 7.8 S was calculated.
for reagins to ragweed /27/. As already. stated, in a number of
recent.studies\it has been suggested that skin,sensitizing anti-
bodies may be identified with 101A-globulinsHowever, it is
worth pointing out that although the major portion of this
/globulin fraction has a sedimentation coefficient of 7 S minor
compOnents with sedimentation cOefficionts Of 10.5 S and 13 S
? are also present in this serum fraction /28/.
Immunochemical properties'
Specific precipitation of serum components present in
,chromatographic fractions possesSsing highest skin,sensitizing
activity with a rabbit antiserum to human Iglobulins resulted'
in the removal of all skinwsensitizing antibodies /29/. However,
'since yIA-globulins,,as well as 7 S and 19 S 'share
common antigenic determinants, these results siftly indicated
that skin-sensitizing,antibodies belonged to the class of itmuno-,
?
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Sehon 6
globulins but did not specify more precisely their antigenic
nature. In a more recent study it was demonstrated that Pemoval
"of,7 S r-globulirls from a skin-active erum fraction by
precipitation with an antiserum, ,rendered specific to 7 S
. .
1:-globulins by prior absorption with T1-globulins, did not
result in a decrease of its reaginic activity /18/. However,
'complete removal Of skinvsensitizing activity from three rag-
weed allergic sera was achieved by absorption with an antiserum
rendered specific for-globulins by the precipitation of
antibodies directed to the determinants of 7 S f-globulins. Thus,
one can conclude that at least some of th6 antigenic groups of
skin-sensitizing antibodies in these three sera were identical
to those of
globuliris- However, since riA-globulins have
,a tendency to complex with other proteins /28/0 and since recent-
ly three sera of ragweed-sensitive individuals with high P-K
titers were found to be devoid of ir glpbulins /26, 30/,
? it is felt that additional eVidence is necessary before skin-
Sensitizing antibodies may be unequivocably identified with
nAHglobulins. It is conceiVable that akinvsensiting anti-
bodies are complex ,molecular species, one of their buildings -
,
blocks being ?1A-globulins. This latter component might be
also responsible for the fixation of skin-sensitizing antibodies
to tissue and for their retention by the placenta and choroid
plexus. In this .connection, it ought to,be pointed out that
.Iahizaka et al. /31/ demonstrated that passive sensitization of
human skin with ragweed reagin could be blocked with purified
normal human 414...g1obulin. fraction as well as with the A-chain
/and not with B chain/ of I1A-globulins /32/, In the light of all
the experimental results derived from ultracehtrifugal, chemical,
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Sehon 9
ohrOmatographic.andmmunoehemical studies, onecannot deny
.the fact:that-there is Ancroasing circumstantial evidence in
,
.favour of reagins belonging, to thegroup-of: fiA-globulins
.with:sedimentation:coefficiente higher than 7.
The very recent investigation of Yagi et al; with the help
of:radioimmtneelectrophoreSisi revoaled that ragweed-binding '
.,capacity of allergic :sera Was not iimited.to a single protein /33/0
.but was associated with.the _ of'all the 8 sera
te-sted and also with-the .1aA4nd/or 71-globulins. Of cdurse,
p these results do not 'prove, that skin-sensitizing antibodies
are directly implicated in this reaction and,-" as will be shown
later; it was actually demonstrated that sera of non-treated'
allergic-individuals possess, in addition to reagind, antibodies
devoid of skin-sensitizing activity but with binding capacity
,
for 'allergen/s/. thus, theselresults-lend,further support to the
evidence presented above that there'exists a spectrum of anti-
bodies 'in allergic' serum differing 'in their physico-chemical
immunochemical properties.
Blocking antibodies
Formation of blocking antibodies can bp induced'in both
allergic individuals and nen-allergic volunteers and, as has
? ? ,
been shown //, the various properties of blocking'antibodies
elicited in these two 'types of individuals are identieal. More-
over, blocking ability is not confined_only to antibodies produced
in Man, bUt is also possessed by antibodies formed by rabbit, .
JT,oat and log 'on immunization with appropriate allergen..
-:The chemical nature of bloCking antibodies resembles that. .
of precipitating antibodies and differs signifICantly from
that of skin-sensitizing antibodies. This, blocking antibodies
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Sehon - 10
alio not inactivated by heating at 56?C, are stable under various'
condi4t.ons of storage andrhandling and are net degraded by
reductiorivith-0.1M mercaptoethyIamine /17/. Moreover, digestion
of blocking antibodies with pepsin, with papain, or with pepsA41
followed by reduction with mercaptoethylamine - reactions known'
to lead to the breakdown of precipitating rabbit antibodies
to smaller.divalent or univalent antibody fragments - resulted
in the degradation of blocking antibodies to fragments which
were shown to retain to ability of combining with the allergen/s-
and of blocking the P-K reaction to the same extent as intact
blocking antibodies /17/. Similarly, fragments prepared from
precipitating rabbit anti-ragweed antibodies behaved in the
same way, i.e. the blocking titers of the fragments were iden-
tical to those of the intact antibodies /17/.
The bulk of blocking activity was invariably found associated
with the electrophoretically slowest migbating serum components,
i.e1 namely with -,2-globulins /4/, but occasionally in oera
containing large amounts of blocking antibodies, a small portion
of the blocking activity was detected also in the faster migration
-i-globulin reaction. This behaviour is similar to that of con-
ventional procipitt4ng antibodies, which have been demonstrated
to be physico-chemically heterogeneous and to extend electro-
phoretically from the region of the slowest r-globulins to that
of ?(2-globulins /34/. In the light of these findings, it seems
reasonable to ascribe the ragweed binding activity of T2-globulins,
,..as demonstrated by hemagglutination and by radio-immunoelectro-
phoresis, to blocking antibodies. Moreover, the binding of 1131_
labelled ragweed pollen constituents by 71m- ands jiA-globulins
/33/ reflects, in all probability, tpo clectrophoretic and
ultracentrifugal heterogeneity of blocking antibodies, though
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Sehon - 11
skin-sensitizing-antibodies may participate also in this reaction.
As demonstrated previously /4, 8/, the sedimentation coeffi-
cient of blocking antibodies was close to 7 S which would also sup-
port, the interference that.blocking antibodies have properties
identical to those of the conventional antibodies produced on
actPe immunization. Similarly, the chromatographic behaviour
of blocking antibodies ,on DEAE-cellulose was analogous to that
of precipitating rabbit antibodies /9 12/, i.e. the major
,pOrtion of both types of antibodies was eluted in the first
fraction with 0.01M phosphate buffer at pH 7.5. Moreover,
in the first few eluates, .i.e. in the first subfraction /Fraction
1A/, which were rich in antibodies, there was no detectable
skin activity /12/. It outght to be also pointed out that small
amounts of blocking activity were found in subsequent chromato-
graphic
fractions eluted with buffers of higher concentration
and of lower pH /12/: Essentially identical results were obtained
with sera of non-allergic volunteers or rabbits, immunized with
ragweed pollen extract, and with sera of non-treated or treated
allergic individuals, except that the first chromatographic
fraction /Fraction 1A/ of sera of non-treated allergic indivi-
duals had to be first concentrated - to a hemagglutination titer
of about 2,500 -.before the presence .of blocking antibodies
could be demonstrated /12/. Moreover, after appropriate concen-
tration of subfractions lA to a hemagglutination titer of the
.order 40,000 precipitating antibodies were readily demonstable by
? the micro-OuchterlOny procedure /35/. On the basis of these
? findings, it was interred that these various immunological
? manifestations were attributable to the same type of antibodies
and, consequently, it was proposed that blocking antibodies
had the properties of normal immune antibodies /14/. On the
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Sehon - 12 ?
basis, of this argument, it must be conCIUded that even non,
"treated allergic individuals.; i.e. Patients who have not received
any hyposensitizatien. treatment', form blOcking.antibodies and,
therefore,.the older elassifiaation of sera of allergic indi-
.
.viduals into two categories, -.depending on whether they were
obtained from patients prior to or after. hyposensitization treat-
ment, can'be regarded as purely arbitrary. It ought to be stressed,
however, that during h7poSonsitization treatment, the patient
? .may preduce additional antibodies to antigenic constituents in
, -ragweed, which may have no relationto the allergenic deter-
. minant groups.-These antibodies would then excapo detection by, the
in vivo procedure fo'r the'domonstration'of blocking antibodies,
,although their presence could be 'manifestedby in vitro methods,
- by hemagglutination and. preeipitation.. Of course, similar )
considerations apply_ also to blocking.antibodles produced
nsp6ntaneous1y" by the non-treated allergic individual, as well
as by non-allergic volunteers and rabbits on immunization.
The relationship of hemagglutinins to skin-sensitizing and
-blocking antibodies.,
From' an analysis of theihemagglutination.and P-K titers
of sera of non-treated allergic individuals, it appears. that,
-although all sera tested were never devoid of-hemagglutfnins, the-
re exists no simple.relationship'between those two titers, i.e.-
sera with high PK titera-may possess low or high'hemagglutination
:titers, and sera.with low P.K titer may possess high or low
? hemagglutination titers. This lack of correlation seems to
suggest that-the homagglutinating ability of sera of.non-__
-.treated allergic individuals is duo. to multiple factor's, the
proportion of which' differs from one serum to another,, and
- ?
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Sehon , 13:,
i'iepresents the Individual variation in antibody reaponse amongst
a group of allergic individuals. The possibility that multiple
hernagglutinins may bejresent 'in allergic sera is also supported ?
by the findings that -he, hemagglutinatton titers obtained with
erythrocytes.send-tiZed via tolylene-214-diisocyanate were sub-
*
atantially lower than the S:DB.4iters.and that there was-no
obvious?relatiOnship between these two sets of titers /36/.
. 'HThere soots to exist some cOrrelation between the.hemag7
glutination and the blocking titers of the ,sera of treated .
allergic individuals /aa well as of immunized non-allergic
volunteers and rabbits/, in as Much as, in general, a detect.,
able blocking reaction could not be obtained unless the serum
/or serum fraction/ had a SDB-hemagglutination titer of about.
2,500; correspondingly, seim with higher hemagglutination titers
had also proportionately higher blocking titers. The fact that
serum fractions possessing blocking antibodies were found to
have:also the other properties of "conventional" antibodies
Produced on active immunization, 44e..precipitation.with the
homologous antigens or. allergens, passive cutaneous sensitization'
- of guinea 'pigs /17/, could be Interpreted as indicating that.
.? either 1. there are multiple types of antibodies Present in
these sera, each type 'being responsible for one of the, immunp7
logical manifestations .or 2.. in accordance with the "unitarian.
concept", .one and the same type. of antibodies is capable of
-
participating in different immunological reactions, Obviduslyl.
!neither of these two. possibilities' can be ruled out as long as '
the serum factors responsible for the different immunological
-manifestations are not isoated in a pure form and.no dose
response relations Can, beelstabliahed for theae reactions.
. . _ . . . . . .
?
?
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Soh= - 14 ?
In order to settle imequivocially the question ?as to
? . . ,
Whether or: not.there:existS any relation between hematglutinins
and skinTsensitizing'and/oz4, blocking antibodies, it would be
. ? .
necessary to establish if 1, the hemagglutinating ability of
sera of non-treated allergic individuals as a property of skin-
sensitizing antibodies or, exclusiVoly,of.a type of blocking
..antibodies pi.oduced "spontaneously",.i.e.. elicited simply by the,
-casual exposure to the allergen/V. and not in response to
hyposensitization treatment; ? 2, the hemagglutinating ability of
seraof treated allergic individuals is a property of blocking .
.antibodies or of a different type of antibodies,directed against
some antigenic deterMinants: of .ragweed pollen constituents which
'are unrelated to the allergenic.greups; 3. blocking antibodies.
?produced on active immunization /in allergic individuals,.in
non-allergic volunteers or in-experimental anitrialsi are iden-
tical to the blocking antibodies produced 'spontaneously" by the
allergic individual or exposure to. the allergen/s/.
\
The similarity between the physico-chemical and chemical
?
properties of blocking, antibodies and of hematglutinins strongly
. suggest that the major. portion of the hemagglutininS of sera of
treated allergic individuals may be identified with. blocking
antibodies. On similar: grounds and also in view of the reactions
of identity/given by antibodies of sera of treated and non-
.
;treated individuals in:agar .gel diffusion experiments /5/, one
:may postulate that blOCking ;antibodies produced "spontaneously"
:by the non-treated allergic individual are indistinguishable from
those produced on.hyposensitization treatmenf. On the other
.hand the lack of correlation between the hemagglutination and
titerSof sera of non-treated - and treated allergic individuals,
.and more importantly the findings that the hemagglutination titers
?
1
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Sehon 15 ?
of chrematographic fractions possessing the bulk of skin,sensitiz-
ing activity represented only a small fraction of the hemagr
glutination titer of the whole serum /12/, indicates that even if
skinsensitizing antibodies had hemagglutinating capacity
their contribution to the overall hemagglutination titers would
be minimal. Obviously; in order to establish if these two dif-
s.
.foreht immunological reactions are duo to the same serum. factor/s/,
it is necessary to isolate the corresponding antibodies in a
pure state, or at last to separate them from other antibodies
? reacting with the same antigen
With this in view a number of studies have been conducted
using different physieo-chemical and immunochemical fractionation
prOcedures. Suffice it to say here that hemagglutinating capaci-
ty was associated with fractions contaihing skin-sensitizing and/
, or blockin antibodies and never with any fraction devoid of either
of those antibodies. From a. careful analysis of the distribution
of the hemagglutinating activity infractions isolated by ultra-
centrifugation in partition coils, it was concluded that at least
two physicochemically distinct hemagglutinins were present in
"allergic sora. and it was suggested-that one of these hemag-
:glutinins ha4 a sedimentation coefficient of the order of 19 S
and that the other had a sedimentation coefficient of 7 S.
On the basis of the similarity of the sedimenting proper-
ties of the formerczlaas of hemagglutinins and of skin-sonsitiz-
.ing antibodies, it was suggested that sera of non-treated allergic
individuals might cOntain two types of antibodies: 1.antibodies
with sedimentation coefficients higher than 7 8 which may 13ossess
? both skin-sensitizing and hemagglutinating abilities and 2.
antibodies of the.conVentional 7 S type, possessing only hemag-
glutinating activity. These latter hemagglutinins may be identi-
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Sehon - 16
fiale With the bulk,of.the blocking antibodies.
The problem of identifying homagglUtinins.with skinsensit-
izing and/or blocking antibodies is, however, tompli:cated since
the sensitivity of the hemagglutination reaction is lower than
that of the p-K test and, -furthermore, the sensitivity of the
P-K test is not a constant value, .i.e. the amount of antibody
detectable by this technique depends both on the antigenantibody
system as well as on the individual antiserum used. In general.; the
SDB-hemagglutination titers of sera of ragweed-,sensitive indi-
viduals wore higher that the titer of grass-sensitive individuals,
both for a group of non-treated and_treated allergic patients
/11/. Correspondingly, the hemagglutination titers of the chro-
matographic fractions containing the bulk of reagins to ragweed..
wore strikingly higher than the hemagglutination titers Of the
corresponding fractions of sera of grass-sensitive individuals.
.Thus, hoagglutination titers of these fractiois obtained with.
spra of ragweed-sensitive individuals were of the order of 10
to 40 /12/,. and not higher than 4 with sera of grass-sensitive'
individuals /10/. This difference in the hemaggldbination titers
c7,
of the reaginic fractions of these two types of sera is difficult
to interpret., particularly since it might be merely due to
the difference in the sensitivity of the hemagglutination techni-
que for the two systems
The fact that skin-sensitizing activity, can be destroyed
by heating without impairing the hemagglutinating ability does
not necessarily imply that these two manifestations are .associated.
with two distinct typos of antibodies. It.is'conceivable that the
same antibody molecule may possess the two-antibody combining Sites
required for he'hemagglutination reaction /and which are com-
plementary to the determinants ? of ragweed pollen constituents/,
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,
Sehon - 17
in addition to the Site which has the ability to become fixed
? tO Akin and tissues. It. can be also visualized that the portion
.of the,moleeule responsible for skinwsensitizing can be destroyed
..,
pr modified irreversibly by heating, without impairing the abi-
lity .
. . , .
of the antibody combining sites' to react with ragweed aller-
? -TheoreticallY,: one may visualize that some of the hemag_
:glutipatihg. antibodies may: be directed against some antigenic
'Ideterminants which,arO.notinvolved. in the reaction with re-
agins and Which, .nevertheless, may be situated on the same
;
molecule in the vicinity of the allergenic determinants, or on
'
?antigenic.constituent6 of ragweed Rollen which aro unrelated to
the allergens. From all these cohsiderations?ohe is' led to 'con-
dude that the contribUtlon of skin-sensitizingantibodies to the.
hemagglutination.titer or allergic sera is +Dilly minimal. Nevertho.
less; the TesultA of all physico-chemical fractionation studies,
fractional precipitation, electrophoresis, ultracentri-
. ? ?
fugation,. chromatography/ do not rule out the possiblity that
skihsensiltizing antibodiespossess.also hemagglutinating pro- ?
!The comparison of the behaviour of skin.sensitizing and
blqpiing ahtilRodies On degradati.on.with:papain /17/ add further
'support to this interference. In these experiments the rate Of .
inactivation, of reagins was measured ih terms of the decreaSe.of
skin-sensitizing titer, whilst the,rateof degradation Of bloek7
';ing antibodies was measuredin terms of the decrease of the
;.hemagglutination titer; thellattor proeddure had to be resorted.
-.to. Ape() it 'had been shown that degradation of blocking anti-
bodies' to univalent 'fragments was not 'associated with any loss
; .
lof blocking capacity. Thus the kinetics of the degradation
; ? -,
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Sehon ? 18
of the hemagglutinins in. sera, of non-treated allergic individuals
with, papain appeared to be,represented by two rate processes':
- -
an initial rapid destruction of about 95 percent of the hemaggluti-
nins' within the first ? 30-60 minutes of digestion, followed by
"a much slower inactivation of' he residual hemagglutinating acti
vity. The first process, by analogy with the rate of degradat-
ion Of blocking ant.Voodies elicited on active immunization, was
considered to represent the degradation of blocking antibodies
produced "spontaneously" by the non-treated allergic individual.
On the other hand, the second proces6, by analogy with the slow
a
rate of inactivation of skin-sensitizing antibodies, was con-
sidered to represent the degradation of antibody molecules possess-
ing both skini.sensitizing and hemagglutinating properties. It
ought to be stressed that, because of the inherent inaccuracies of
the methods used for the quantitative determination:of reagins
and hemagglutinins, these kinetic data could not be subjected
to a mathematically rigorous treatment to test this interpretation.
It is, therefore, nOt possible at this stage, -Co dismiss'the
alternate explanation, namely .that the apparent similarity
between the rates of enzymatic degradation of skin?sensitizing
antibodies and of a small portion of the hemagglutinins of sera
.of non-treated allergic individuals might be purely fortuitous
and might reflect simply the presence of multiple and chemically
different hemagglutinins in these sera.
.Additional evidence' that each serum contained multiple
hemagglutinins was derived from studies in which increasing volu-
: mestof allergic sera were absorbed with a constant amount of a
specific immunosorbent /13/; T4e results of, these experiments
/?
demonstrated that the.finer-detalls of each of the absorption
.curves representing the absorption Of hemagglutinins and/or,
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Sehon
gins !froth serum faction obbainod'by chremategraphy on ?
DEAE-cellulose Were :peculiar to the serum used. In addition, the
-removal-of skin-sensitizing activity from Fraction 7 obtained
" by chromatography on 15EAE.-celluloae /i.ej the 'fradtion contain
in the major portion of reagins/vot each of these two sera
was almost parallelled by the removal of the hemagglutinins.
,
'These results were interpreted-ad indicating that the.hamag-
. .
capacity of Fraction 7 was an inherent_property.
1
of skin-sensitizing Antibodies, namely that reagins were either
antibody molecule Or molecular 'complexes .possessing /at leas/
two sites eapable of combining with tho allergen, in addition to
.the site which was. capable of .p.ttaohing itself to the skin.
?Furthermore, it was eholn.that coneiderable amounts of skin-
,
sensitizing antibodies could be displaced by blocking anti-
boclies.from/an immunosorbent'which had been previously saturated
with respect to reagins.. These results indicate clearly that
allergic sera contain .a spectrum of antibodies -with different
?.
physico-chemical 'and biological properties and which are directed
against antigenic groups on the same molecule/s/ of the ragweed
conetituents.
In,summarypit'can. be stated that in spite-of the accumu-
-lation of a large amount of.information,on the physico-chemical
'and'immunoctexioal properties of skin-sensitizing, blocking and
)1emagglutinating,antibodies, the nature of 'the serological factors
\
involved in the appropriate immunological Manifestations has not
been unequivocally established and no simple relationships have
boon derived' amon6st.them., However, it is obvious that the,
antibCdios in allergic zera are heterogeneous with respect
to,. their size, shape, composition, Charge,?affinity-for the
-
.
4-
E.? .inl n U
P ? R. GrftuiSE ONLY
,
, -
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Sehon.- 20
- antigen/-/ ad ability to 'become fixed to tissues. 'Nevertheless,
it seems* plausible ,to conclude that blocking antibodies 'may be
;regarded as Anununoglobulins of the conventional "/ ..type 'with
/2
a sedimentation coefficient of. 7 ,S, Which are.. detectable by
classical immunological techniques. On the other. hand, skin-
sensitizing antibodies are electrotthoreticaliy faster-moving
globulins belonging possibly ;to the class of
and/or i_globulins, they have sedimentation coeffi-Aorat/s/
higher than 7 SI and may -contribute to a small' extent to the
hemagglutination titer of allergic sera.
. ??/
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; Sehon - 20
\ antigen/s/ and abill4i-to pecomV fixed to tissues. Nevertheless,
it seems plausible :0'conclude that blocking antibodies may be
regarded az immunoglobulinsof'the conventional :,-,-type with
? ' ? ?
- 2
a Sedimentation toeffiCieht of 7 S.) which are deteCable by
? classical immunological techniques. On the other hand, skin-
sensitizing antibodies are electrophoretically faster-moving
globulins belonging 'possibly to the class of.lriA-globulins
and/or elim-globulins, they have sedimentation coefficiont/P/
higher than 7 3, sand may contribute to a small extent to the'
hemagglutination 'titer of allergic sera.
?
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?
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s
r,
. Sehon
?
Properties of antibodies in allergic sera
/as summariied in 1.959 /8/.
A
Reagins Blocking antibodies
does fix to skin does not fix to skin
? does not pass .through placenta.. passes through placen-
ta
heat labile with respect to skin-
sensitization ' heat stable
.combines with Ag in vitrocombines with. Ag in
vitro :
. .
:.does not precipitate at 30% -
_ammonium sulfate
precipitates at 30%
ammonium sulfate
r1-/or 4-/globulins by electro-
phoresis
. 7b-globulins by
electrophoresis
4111????????=1...?111?1
fraction III by Cohn 's method fraction II by Cohn 's
method
'sedimentation -constant 7 S sedimentation constant
= 7 S
Both sera of non-treated and treated individuals, contain-
ing skin-sensitizing and/or blocking ,antibodies, possess hemag--
:glutinating
?
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?
. References
Sehon - 22
a. Prausnitz, G. and Kilotner,.T.t.-Zb1.Bakt.0 Parasitenk. 86:
,160 /1921/.'
2. ? Cpoke;:R.A., Barnard, J.H. .Hebald, S? and Stall /A.:
,J.Exp.Med. 621733 /1935/6'
Boyden, S.V.: In. "Mechanisms of Hypersensitivity" p.195
. eds..
P.H.Shatfer G.A.LoGrippo and M.W.Chase/ Little Brown and
Co.', Bo.ston.1959.
? 4. Sehon, A.H.t.In "Mechanisms of Hypersensitivity" p.61
eds.
/RT.H.Shaffer, G.A.LoGrippo and M.W.Chase/ Little, Brown and.
Co., Boston 1959.
' 5. Lawrence, H.S.: In: "Cellular and Humoral Aspects of the
Hypersensitive States" p.279 /S.H.Lawrence, ed./. Hoerber-
Harper, Inc., New York 1959. .
?
6. Middlebrook, G. and Dubos, R.J.: J.Exp.Med.88:521 /1948/.
7. - TurCotte, R? Freedman, 8.6. and Sehon A.H.:_Am.Rev.
?-Resp.Dis. 88:725 /1963/.
8. Sehon, In: "Mechanisms of Antibody Formation, P,791.
, /M.Holub and L.Jaroskova, eds./ Pul?lishing House of the
Czechoslovak Academy of Sciences, Prague 1960. ,
9-0.' Augustin, R.: In: "Mechanisms If Antibody Formation", p.94
/M.Holub and L.Jaroskova, eds./ 'Publishing House of the
Czechoslovak Academy of Sciences, Prague 1960.
? 10.
11.
. 12.
'Augustin R. and Hayward, B.J.: Immunol. 3 :45 /1960/.
,Frick, Gyenes, L., and Sehon, A.H.: J.Allergy 31:
216 /1960/.
Perelmutterl:L., Freedman,. S.O., and Se-Wen, A.H.4 Int.Arch.
Allergy.19:129 /1961/.
Perelmutter, L., Freedman, S.O., and Sehon; A.H.: J.Immunol.
.89:623, /1962/.
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Sehon - 23
14. Sehon, A.14: "Allergology" p.300 /E.A.Brown, ed./
:Pergamon Press, London 1962.
.15.; Sherman, LB.: In: Proc.3rd Internat.Congr.gf Allergology
p.155 /B.Iyialpern and A.Holtzdr, eds./Editions Medicales
jaammarion, Paris 1958.
16b!, Stanworth, D.R.: Brit.Med.Bull. 19:235 /1963/.
17.' Gyenes L., .Sehon, A.H., Freedman, and Ovary; Z.:
Int.Arch.Aliergy 24:106,4964/.
;B.' Fireman, P., Vannier, W.E., and Goodman, H.C.::.J.Exp,Med.
117:603 /1963/.
19. Sehon, A.H.: In "Proceedings of the Third Congress of Al1ergo-
? logy",.p.209, /B.N.Halpern and A.Holtzer,eds./ Editions
? Medicales PlamMarion, Paris 1958.
20 Stanworth, D.R.: Immung1.'2:384 /1959/.
21. Gyenes, L. Gordon, J., and Sehon, A.H.s'Immunol. 4:
177 /1961/.
22.: Terr, A.I.:and Bentz., Ped..Proc. 23:402 /Abst.1806/,
/1964/.
23. Heremans, J.P. and Vaerman, J.P.: Nature 193:1091 /1962/.
24.. Campbell, D.H. and Sehon, -A.H.: In "Mechanisms of Hyper-
sensitivity" /note on p.120/ P.H.Shaffer, G.A.LoGrippo and
M.W.Chase eds./ Little, Brown and Co., Boston k959. -
25. Rockey, J.H. and Kunkel,\H.G.: Proc.Soc.Exp.Biol. and Med.
110:101 /1962/.
, 26. Loveless, M.H.:,Fed.Proc. 23:403 /Abstr.1811/, /1964/.
27. Andersen,: B.R. and Vannier, Fed.Proc.,23, 402 /Abstr.
1807/0 /1964/e
28. Heremans, J.: "Les Globulines Seriques du Systeme Gamma, Leur
Nature et Leur Pathologies". Masson et Cie, Ed., Paris 1960.
'
.9
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Sehon . 24
29 AUgustin, R.:, Acta, Allergol. 16:473/1961/.
, . 304 Arbesman, C.E. /personal 'communicatiOn/; ROckey, J.H.
,
/personal communication/...
^
51. Ishizaka, K4; Is,hizaka, T4, and HornbroOk, M.M. J.Allergy
-? 34:395 /1963/. .
32. Ishizaka, K4, Ishizalea, Ti-, and Hathorn; E M.':Fed.Proc.
23:402 /Abstr;1809/, /1964/.
3, :Yagi, Y., Maier, P.,. Pressman, D., Arbesman, C.E., and
Reisman, R.E.: J.Immunol. 91:83 /1963/.
34. Webb, T. and Lapresle, C.; J.Exp.Med. 1141 43 /1961/.
Perelmutter, L., Lea, D.J., Freedman, S.O. , and Sehon, A.H. :
Int.Arch.A4ergy 20:355/1962/. ?
,
36. Lo003, L. and Sehon A.H. Immunoch_em. 1:43 /1964/.
?
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.1
:For. 'discussion in the ',t4ialI,1 C.
- .
'. A- Method for The'Isoiatibn,Cfl-lobulin-from Human Serum
Using salting-out. and Chromatography'
,?.
r
.F.Heremans,:A.O.CarVonara and Giuliana Manci.4
.;
Department of Internal Medicine A, University Clinic St.Pierre,
?
Louvain, Belgium
H
:.Abstract
IgA/ may-be isolated .
?!
from normal or pathological human sera by a series of steps
, ?
, consisting in the removal of euglobulins at pH 6.5 and pH 5.4, ?
alting-out of the globulin:fraction by means of 2 M ammonium
1
sulfate at pH 6.8r? chromatography of .carboxymethylcellulose,
. and chrdmatagraphy on-triethylaminoethylcellulose. The end-
product shows no traces of contamination with the other immune-
globulins /7 S rand 19.S r./. ?
? Y.A. method for the isolation of- from'hunian.serum
using Salting-ont and chromatography. ,
The iMmune globulin called IgA/.
.:was initially prepared. by Means of 'aprocedure involving preci-
AAtation with- c sulfate,salting-out.with amm6nium?sulfate, ?
,and preparative.elctrophoreSis /1/. In spite of different impro-
vements./2, 3/1 this method hits proved 'to be poorly, reproducible
and to yield products Whose purity was often unsatisfactory,
.Contamination with trace amounts of 7 S and .19 S
?.,globulin has been particularly inconvenient, as the isolation ?
? .
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(:12 from human serum is usually undertaken with the,
:11.im of assessing-its antibody activity spectrum. Chromatographic
. methods have given excellent results when applied to the isolation'
I 1
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[UHLduitL
Heremans et al. - 2
of 21A- type myeloma Proteins /4/ or to the purification of the
.G'111:-g1obu1in:present in pliik /5/. However these methods have
proved much less Suttable for the recovery of q1A_globulin from
normal serum, in spite of some claims to the contrary /6/. The
difficulties/in obtaining turified r1-globulin from such a
complex a mixture?of proteins as human serum have been stressed
by Davis, West and Hong /7/, Who investigated several fractionat-
ion procedures.
,The method to be described here, permits the isolation
of highly purified 'r1A:- globulin.frOm normal human serum. However,
.yields of purified protein are still low.
Step 1. Removal of euglobulins
? The aim of this step is/to remove two proteins which would
be difficult t6: eliminate at later stages of. the fractionation.
:Oneis r1-macroglobulin-/19 ur/, the larger part of which is
:removed by dialysis against dilute.buffer of pH 6.5. The other
? euglobulin is ()." 1A- / and ?lir/ globulin, which precipitates during
'dialysis against dilute buffer of pH 5.4.
One volume of serum is diluted by addition of 9 volumes of
,0.02 M NaH PO /Na HPO buffer of pH 6.5 and dialyzed against
. 2 4 2 4
500 volumes of the same buffer, with cOntinuous stirring. The
dialysis.is performed at 40 and the outer buffer solution is
renewed twice over, a period of 84 hours. The euglobulin precipitate
.is removed by centrifugation.at.3 000 x g during 10 minutes, at
room temperature.
The supernatant is now dialyzed against 50 times its volume
01.0.02 M NaH2PO4 /Na2HPO4 buffer Qf pH 5.4, with continuous
stirring,. at 4?..The.outer bilffer is renewed twice over a period
'of 24 hours,. and the precipitate. is eliminated by centrifUgation
DFFICFAL VSE ONLI,
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?
Heremans.et,al. -
? ?at:3000 g dUring 10minutes:, ,a .room temperature.
.Step 2:4 .Salting-oUt af'the Crude immune globulin_ fraction by.
t .--, ?
menns of 2-M ammoniumisulfat
. . .t. .
,.
. . - .
.? . ? .
:Thin?Step_is aimed at 'tT.L.eremoval 9f-the bulk of the albumin
-mid Other. irrelevant protein.'
?
.The supernatant.from the 'second centrifugation is mixed
, with-'an equal volume of a 4 M Solution of ammonium' sulfate: This
.solution hab its H adjusted to 6.8, before use, by means of the
requir.red amount of a saturated NaOH. solution. After mixing the
.protein solution with :the ammoniUm sulfate.; the PH is checked
and, :if necessaryl-adjusted?to 6.8. After 1 hour incubation at
room temperature /20?/2 the precipitated globulins are recovered'
?
by centrifugation at 13,000x g for 10 minutes, at room temperature,.
The precipitate is redissolVed in distilled water, employed in an,
amount equivalent 'to 10 times the original sertim.volume. The protein
, solution thus obtained is 'precipitated again by adding its ovii
volume of the' above dosCribed 4-M ammonium sulfate solution.,; The
new precipitate-is left to stand?fOr 1 hour, at root temperature,
,and Centrifuged off at, 13,000 x g; for 10 minutes, at room
-*,teMperature. The final, sediment is redissolved in 10 times the.
;original serum volume of dietilled'Water,
. ?
.step 3, Chromatography on'eyrboxymethYlcellulose /CM-cellulose/.
?
. The aim' of this 'step is to free the preparation from all
.but traces of 7 S
.?The chromatography column'iS packed with an amount of
carboxymethylceliulase /Serval Heidelberg,. 0.68 mEq/g/ calculated
t ,
,41.1. the basis of 0,5
serum sample. Prior'
days by n_tirriiig in
, ?
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g' of CM-Cellulose per ml of the' original
?
to Use the CM-cellulose is washed for several '
. f
'approximately 10 times. itn awn volume of 0,25
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' ?
,
,
Herertans ot al, 4 ,
?
N NaOH solution, then with portions of distilled water until
the? pH of the washing ,fluid. ceases to be alkaline; ana finallY
with several portions ,of McIlvaine phosphate-citrate buffer of
pH 6.0. This buffer is made by mixing 48.5 volumes or 001 u.
citric rid and 51.5 volumes of 0.02 M Ha2H?04 . 2H00. the final
suspension j3 poured into the column and allowia te SOditint,
after which the column is waihed with 50 ml of thi Weide bUffeV
per gram of CM-cellulose employed.
The sample to be chromatographed is dialyfed against 50
times its own volume of the same buffer, at 40A The outer fluid
is renewe4 twice during the 24-hour period. of dialysis. Some
prozipitate will form and is removed by centrifugation, after
'Which the sample is concentrated to one.quarter its original
volume, by mains of ultrafiltration in vacuo. The concentrated
sample is now applied to the chromatographic column and recoved
,in the effluent by washing the column with the same buffer, at
room temperature, until the extinction at 280 M/u of the effluent
drops to the baseline value,
Step 4. Chromatography on triethylaml.noethyloollUlose /THAE-
cellulose/.
Thi; step is Intended to separate the 1..gLOtlin ?from
the accompanying psroteins mainly ot-globulins and albumin, which
,are also present in the breakthrough volume from the CM cellulose
column.
? The chromatography column is packed with an amount of trio
ethylatinoethyleellulose iterva, Heidelberg) '0468 mtq/a/ cal*
,OUlated on the basis of 2.0 g of THAEwsellulose per ml, of the
original seUM sample. Prior to use the TkAt*cellulose is washed
for several days by stir*ing in approximately its own Voltun? o
of 0.8 R 240H, and then with seveVal por,tions ot 4iiitilled water
U ti
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Heremans et al. - 5
until the pH of the washing fluid 'ceases to be alkaline. After
decantation of the final bath of distilled water, the slurry is
resuspended in 10 times its:volume'of 0.2 M NA2HPO4 . After one
.hour the buffer is removed and replaced by several portions' of
distilled water until the,pH cif the washing fluid ceases to be
alkaline. The wet slurry is resuspended in 10 times its' volume
of 0.2 M NaH2p04 and, after one hour,, washed again repeatedly with
distilled water until the pH ceases to be acid. The paste is now-
suspended.in lo times its volume of 0.01 M dis odium' phosphate
buffer', Of pH 8.0. This buffer is, obtained by mixing 50 volumes
.of 0.01 M Na2HPO4 and-3`volumes of. 0.01 M NaH2PO4. The final
suspension of TEAE-cellulose is poured into the column and
allowed to aettlel.after whi4h the column is washed with 50 ml.
of equilibratirig buffer per gram of TEAE-cellulose employed.
.The sample to be chromatographed is dialyzed' against 50 times
its awn volume of starting biefer /0.01 M; pH'8.0/, at'4?. The
outer fluid is renewed twice during' the 24-hour period of dialysi.
The sample is then concentrated to one-fifth its volume by ultra-
filtration in vacuo, .and run into the column. Five times the
cplumn volume of starting buffer is now passed and the effluent
:discarded. No protein is recovered at this step. Elution is
'now performed with 0.02 Mdisodium,phosphate buffer 'of pH 8.0,
and the effluent is Collected in 3 ml. aliquots. Extinction readings
are mdde at 280 m/u and'eldtion is continued until baseline values
are again reached. This eluate will contain a mixture of 9"
- 1A-
globulin
and'7 S. 7"-globulin in the proportion Of 2 : 1. No
? attempt has been made to pdrify this preparation any further. Elut-
ion is continued with 0.03"M disOdium phosphate buffer of pH
?
8.0 and the effluent is again' collected in 3 ml. aliquots until
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FU
OFFICIAL USE Vij
eremanP et al. -
6
,the extinction at 280 tryu, has returned to baseline values. This
.-..procluot has proved to be dtilic,globulin free from any contaminants
detectable by immunoelectrophoresis and immunodiffusion against
polyvalent, anti.? SI, or anti-19 Sr antisera,
laution.is reeuMed with 0.05 M,disodium phosphate buffer
of pH 8.0 and continued until the efflUent no longer contains .
,any protein. This product still contains much r1A- globulin
together with approximately equivalent amounts o ? -globulins.
.Separation can be achieved by means of preparative eleettrophore-
sis, which will thus yield an 'additional amount of pure 711A-
globulin.
Results and discussion
'Steps 1 and 2 may cause losses of riA-globUlin amount-
ing to as much as 30% of the quantity initially. present in the
serum sample. About 60% of the amount of 2411A introduced Into
the CM-cellulose column are collected with the effluent. From
this recovered 'amount about 28% is-eluted with the 0.02 M buffer,
16% with the 0.03 M buffer, and 5% with the 0.05 M buffer. The
yield of the method is thus about 7% if only the product eluted
at 0:03 M is recovered, and this can be raised to 8.8% if the
riA from the 0.05 M step is\ included.
One advantage of the presently, described method in compa-
rison with the earlier zinc sulfate fractionation procedures
/11 ?2,-3/ is its excellent reproducibility. Another considerable
advantage resides in the purity of' the
rill fractions 'thus
obtained. Such products should prove most suitable for investigat-
ions on the antibody activities of c,?1A-
globulin.
,L
Native 11A-globulin, as also the 7 S T= and 19 S 1'
components of the' immune globulin system, consists of a hetero-.
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' k
HereMans et al - 7
)
genedua..pOpulation of:molecOles which aro distributed,over,a rather
bzoad slectrophortic :spectrum of mobilities. One of the.dis-
, advantagesrof the preSontlydescribed.method is that the purest
Tilfractions hpre.'obtainecl'appear to contain a predominant
-propOrtion of the fastei, component of riA.fatily.. The Slower
portions are elted with the :p..02 M fraction, which, as already
dtated, is heavily :contaminated with 7'S V.globulin.
References
- .
1. R:ereMans, 3.F.,? Heromans, M.Th., and Schultze, 1i.E.2..
Isolation ane-desriptiOn of'a few properties of the.A2A_
:globulin of human seruM.:Clin.chim.Acta,42''96 /1959/..'
.jieremans; !tes globulines seriques du Systeme gamma.
J:eur nature et lour pathologie. Aracia, Brussels; Masson,
ya.erman, j.P., HeremanS, J.F.,and Vaerthan,
C.2 Studies
of the ithmune globulins of human serum'. I. A method for. .
? the simultaneous isolation of the three immune globulins
ruA andrPIA/ from individual mall serum samples.
J.Immunol. 91; 7'/1963/.
? Fahey, J.L.2.Chrotatographic studies of anomalous
and macroglobulinemic serd..J.biol:Chem, 237:
440 /1962/.
Xontreuill? Chosson, A., Havez,7??.and Mullet, 5.1
'Isolement do la 1...A-giobuline du lait de femme. C.R.Soc.,
154::732 /1960/.
- Havez, R.,,Sautipre, Mea.nd Biserte,, G.: Comparaison des -
Methodes de preparatioxx. f.j:2A".g1 obUlines du serum humain.
C. R. Soc.Biol. 156:1641 /1962/.
I
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' 4
Heremans et al, - 8
Davis, N.C.., West, :C.D. and Hong, L..: Purification on 14) 2A"
globulin from human plasma. Fed.Proc. 21: 76 /1962/.
'
? 1
. .
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?
VI
e?',7.- ? 7T ' ' ;
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- tilli liftiWAL-1,14k U1LY'
? :
7,
,
Th Formation(Of Specific 7-S and Macroglobulin Type Antibodies
in Chickens
;-
.14Aiha
. Institute of MiCrobiology, Czechoslovak Academy of Sciences17
'Prague,-Czechoslovakia
?
. : ?
The-heterogeneity!of. antibodies has now.been'demonstrated
.;i:n different characteristics or the antibody Molecule, i.e. in
?
serological properties, physicochemical charaCter and in the
:antigenic :structure /6/i 'Considerable data have been ?Collected'
H *on the formation of different types of' antibodies. For examPle,
-
the immunization of rabbits with foreign erythrocytes lea to .
-the formation of antibodies which were:mainly\of the macroglo-
bulin type at the beginning of immunization, while after repeated
i immunization a large proportion of:lantibodies was formed with
.an electrophoretic mobility of gamma2 and a-sedimentation
constanis of S /15, 14, 8/,. This time courSein the-formation
. of: antibodies. of the gammaim and gamma2 pype was demonstrated
in further, types of corpuscular and 'soluble protein antigens
These facts, pointing to the interrelationship between the
'formation of macroglobulin and 7 S antibodies,, were-further con-
firmed by' the finding that both types of antibody are also formed
together in the lOwer.vertebrates /16, 17/ and.bytthe fact that
a similar time course in the formation of the twb types of anti-
. b9dies was found in the period ofimmunological immaturity /13, 11/.
, Little is -'yet, known of the serological properties of different
'types of. antibodies It has been shown that macroglobulin anti-
.bodio-s have higher haeMolytic activity and bind less complement
. , ;
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FOR ECM USE ONLY
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'Aiha.-.2'
than17-S antibodies /14, ,E3/. Macroglib.bulin,antibodies are also
,
.ess4atially more,effeetiveilitha haemagglutination reaction
.inwhich'.700 times. leSs.macroglobulin than 7 S 'antibodies are
necessary to produce the same effect /9/. These re'sults,
however) touch onlonly.a 'small part of antibody activity and
it would-be rant impOrtant to carry out a comparison and
?different Aerological manifestations on reaction with antigen.
We ha've attemPted to make a comparisop?of the?activity an.,
Specificity of antibodies of the macro81obulin and 7 S?typs.
ChiCkens were used.ds.the source of antibodies since they are. .
very good producers of.antibodies,tO soluble proten antigens
and produce macroglobulin antibodies it relatively high amounts
/11 4/. 1n addition antibody formation in chicken is Very rapid,
a high level 'appearing even after primary' immunization.
A group, of chickens /Leghorn, weight .1.5 kg/ Were-immUnized
'. with:one 'dose -$pfof human serum albumin p-azobenzoic Acid
. .
/p.=ABA-HSA./9/.'
:Antibodies to prOtein carrier 'determined by the haemagglu-.
? i:tination of HSA.sensitized erythrocytes appeared in all chickens
.as soon as On the third day,' after immunization, whereas anti.-
,
bodies to hapten, also .determined by haemagglutination, did not
appear until the seventh day after imMunization and then only'
. ?
'in.low.tities similarly as deseribed.by?Gold and Benedict ./7/.
The birds were exaawulnated on the seventh day and the types
,of present in .the serum determined by ultracentri-
*.fugation in a sucrose. giadient and by haemagglutination of the
:separate fractions, InAdcord with. the. data it the literature
/11, 4/ antibodies to HSA?were of.both the macrogiobuiin and
S typetOn the other hand, antibodies to hapten. were exclusi-
:vely Of the mabrOglobulin type' /Figsl/s-
??
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?Aiha -23
,?
:Since 'in the first ..experiment we ,found. that,: after primary
immunization,.antillapten antibodies were .of the 19 'S type, in
?a further experiment we attempted'. to determine whether anti- ,
, ?
'1)Odiea of two physico-chemical types can be formed to. one
determinant group, i.e. haptenic group bound bound to .the Protein.
molecule. We ? therefore investigated whether antibodies Of the
7 S type were not also; formed -against hapten on repeated immunizat-
' ion.. A further group of chicken's was immunized 'repeatedly with
,)
?: 40 mg. of p-ABA.41SA given intvavenously at weekly 'intervals.
:''Blood was collected On. the seventh day after immunization before
'giving the next "immuniifation close.. As evident from Pig.2,
,bodiei to hapten in the first two collections wero _of the maicro-
' 'globulin-type and after the ,third 'immunization dose a.ntihapten
r.
antibodies. of the 7 S type appeared, so that after further immu
nization antibodies of :both types were present ? in. the serum.
'These antibodies to Jiapten were deqonstrated by haemagglutinatiOn
.of p7ASA,. bound by azo-.linkage to. .erythrOzYtes. In this system,
in addition to the actual hapten, the amino acids, with which
.diazotized p-ABA's 'reacted, , could form part ? of the determinant,
group, whereas. the other carriers,..-.i.e,. HSA on immunization and
erythrocytes on deteCtiOn? are 'quite' different, This denotes
-that both types_ of antibodies*, macroglobulin- and 7 S, are formed
.against, the same determinant group ?and that. hapten, alone or
possibly a haP.ten-azo-amIno acid -residue /12/ are sufficient in'. ,
'both cases to produce a positive reaction. Bauer /3/ demonstrated
19 S and 7 S antibodies to the same hapten ln. rabbits, similarly.
. That the two types antibodies have . the same specificity.
does net, of course, denote that both. must have the same immuno;.
chemical .propertiesi i.e. .an equally large combining site, the same
space Configuration of the coMbining site and therefore an equally
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_firm binding with antigen. Unfortunately, we could not used ,
antiliapten antibodies to Obtain more details abqut macroglobulin
combining site because these antibodies pccured in too small
.'amounts in the serum and attempts to concentrate them were un-
suCcessful. In further -experiments, therefore, we used antibodies
.to BSA which were formed after immunization in sufficient amounts
for serological study. .
The anti-BSA?antibodies were separated into macroglobulin and
.7 S types by filtration on Sephadex G-200. Antibodies from the
?:corresponding fractions were concentrated by precipitating the
globulins with 30% saturated sodium sulphate, and dissolved in
,a small volume of saline. The sodium sulphate was removed by
filtration on Sephadex G 2.
We worked with pooled sera frorn. three groups of chickens,
i.e. taken after primary immunization, after secondary immunizat-
ion and finally with hyperimmune serum. The birds wore immunized
'intravenously with 40 mg BSA ab monthly intervals, hyperimmune
serum was collected after the fifth, immunization.
In accord with the data in the literature, in all three groups
.we found haemagglutinating antibodies in both the macroglobulin
and 7 S fractions. Fig.3 shows the eluates of the primary and
? t
hyperimmune sera from the Sephadex column and haemagglutinating
? titres of the Separate fractions. It is evident that in the primary'
seta most haemagglutinating activity, was present in the. fraction '
containing macroglubulin and in the fraction containing antibodies
of the 7 S type anti-BSA antibodies were demonstrated only after
? concentration with sodium sulphate. In hyperimmune sera haemagglu-
tinating antibodies were present in both peaks.-A most interesting
fact was discovered on' determining the presend)of cross-reacting_
?antibodies'. If a comparison is made of the titres of haemagglu-
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Aiha -"5
tination of erythrocytes sensitized to BSA and cross-reacting
with-NSA in the separate fractions.of,the eluate, it can be seen
thatlahtibadies.cross,reacting with HSA are mainly present in
the fractiohs containing the macroglobulin antibodids; whereas
.in tho 7 S fraction their titre. is incomparably lower as
compared with.thatof anti-BSA antibodies.
?
,Antibodies were further determined by quantitative preci-
pitation. In concentrated fractions of antibodies of 7 S or
'
.'macroglobulin type the precipitin/ reaction was made in. 0.15 M NaCl
and in 1.5 M NaCl 'to determine both types of antibodies /4/. ,
In concentrated. antibodies of the macroglobulin type we were not
successful in demonstrating the presence of detectable amounts
of precipitating antibodies in any of the pooled sera tested. How?.
ever, in the case of macroglobulin antibodies the sensitivity of
the reaction was greatly decreased since these antibodies pre-
cipitate spontaneously so that the control values were so great
that ? they Aid not permit the determination' of amounts of antibody
of less than 20730 /ug Ab/m1. ,
The precipitation of antibodies of the 7 S bype was posi-
tive in 1.5 M NaC1 in all three pools of sera investigated. In
'
0.15,M NaCl the reaction was positive after the secondary Immuni-
zation and in hyperimmune serum, but not In the primari serum.
The amount--of antibodies precipitating in 0.15.M NaC1 dr in M
-NaC1 varied so that the relatively highest amount of antibodies
precipitating BSA at 0,15 M NaCl in'thomixture of hyperimmuno
sera?similarly*as was described by Behedict.et al. /4/. ;This
also, explains the increase in haemagglutinating antibodies in
fractions corresponding to antibodies of the 7 S 'type during
' immunization, Since only antibodies precipitating in 0.15 M NaC1
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Blum:a
-Alha -,6
/4/ are effectim in haemagglutination.
A comparison of the amount of type 7 S antibody Precipi-
tating in 0.15 M NaC1 with haemagglutination titre of that
-fraction showed that the sensitivity of the, haemagglutination
reaction is- within the tisual range of sensitivity for passive
-haemagglutination. If we compare the high haemagglutination
- titres of antibodies of the macrogiebulin type with the negative
results of quantitative precipitation /what means that the amount
? of these antibodies was less than 50./ug Ab/m1/0 it is evident
,that the: macroglobulin antibodies in chickens are much more
active in haemagglutination than the 7 S type antibodies. Evident-
ly in passive haemagglutination chicken macroglobulin antibodies
react similarly as rabbit antierythrocyto rM antibodies, ithere
I
Greenbury et al, /8/ found that for producing .the same haemato-
,
. lytic, effect an incotparably smaller amount of maoroglobulin
,antibodies is needodthan .in haemaggiUt,matiOn with 7 S antibodies.
We next attemted to determine the specificity .and binding.
,power of haemagglutinating antibodies. of the two types., For this,
pUrpoSe we used cross reactions with ESA and inhibition of haemag-
utination. Inhibition was studied by adding 5 /ug of BSA or
'HSA to each tube of serial dilution of' the sera tested and by
comparing the result of haemagglutination with that of the control.
Fig.4 gives a comparison of the reactions of all three" test
sera. It is evident that in cross reactions the antibodies of the
, ,two types differ considerably. Antibodies of the anti-BSA macro-
globulin.type give crossreactions with HSA sensitized erythro-
'cytes to a high degree in all three cases. In the case of anti-
bodies of the .7 S type cross ? reactions were essentially less,
1.e.'these antibodies showed a faib higher specificity to BSA.
Similar differences were olUv1(4.74?in t1,4o 491mpagglutination in-
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)kiha - 7
hibition reitction,,'Macro1310bulin antibodies were only inhibited
by homolOgous antigen to s?small extent, whereas the.reaction
'of the 7 Stype antibodieswas almost completely inhibited by
homologous'antigen. On comparing the results of cross reactions
of macroglobulin :antibodies from .the samples tested no essential
'differences could be found; 7 S type haemagglutinating antibodies
on the other hand cross react in hyperimmune and secondary sera
leas than do antibodies in the primary sera.
The high degree of cross reactivity of macroglobulin anti-
bodies\ can be explained in two ways. First, by a different me-
.
chanisM of formation of these antibodies and' thereby a different
degree of response to separate determinant groups of antigen
used for immunization, which could lead to a higher formation
of antibodies to-determinant groups common for related antigens.
The second possibility explanation is that the combining site
of macroglobulin antibodies is leas complementary to the deter.
minant group of antigen which would permit a higher degree of
cross reactivity. The ,lower degree of complementarity could
be due to the combining sitebesimnaller in macroglobulin antibodies
than in 7 S antibodies or to its being less exactly delimited
in space /less rigidity of the structure of the site of linkage/.
If the .second explanation of the high cross reactivity of macro-
globulin sera, i.e.differente in the combining site of antibody
.were true, this property would necessarily manifest itself' in
greater dissociation of the antigen-antibody complex. This
corresponds to the results of the inhibition reactions which
- show .that the system macroglobulin antibody-BSA.erythrocyte
is only very little inhibited in the presence of free antigen
. which is evidently due to the high.dissociation of the antigen-
antibody complex.
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Aiha - 8
. Summarizing our results we can say that macroglobulin
antibodies have different properties from 7 S 'antibodies. Both ty-
'Pes of antibodies can arise to tho, same determinant group but
the specificity and ?firmnes6 ofethe binding of macroglobulin
antibodies is lower than that of 7 S antibodies.
References.
1. .Banowitz, J., Singer, S.J., and.Wo1fe,,H.41.t.J.Immunold 90:
399 /1963/.
2. Ba?er, D.C, and StavitskY, A B,: Proc.Nat.A.cad.Scl. 47:1867
? /1961/.
3. Bauer, D.C.: ,J.I;Timunol. 91: 323 /1963/.
4. Benedict, A.A., Brown, R.J., and Hersch, R.T J.Immunol.
) 90; 399 /1963/.
5. Benedict, A.A., Hersch, R,T., and Larson, Ch.: J.Immilnol.
91: 795 /19'83/,
6: ' Fahey, J.L. Adv,Immunol 2 41 /1962/.
7. ' Gold, E,F. and Benedict, A.40:4 J,Immunol, 89: 234. /1962Y.
8. . . Goodman, H.S. t J.Inf,Dis.105: 69 /1959/.
9. , :.Greenburg, CCL,, Moore, D.H.0, and Nunn, Immunology
421 /1963/.
10. , NisSonoff, A.- and Pressman, D.: J.Immunoi. 80: 417 /1958/.
11. 'Piha, I.: Symposium "Mechanism of immunological tolerance",
,:Prague 1961.
_ 12. R,iha, I. and Svi6u1is, A.: Folia microbiol. 9: 45 /1964/.
'13. . Smith, R.T.: In Ciba Foundation .Symp.!"Cellular Aspects of
Immunity"? Churchiil? London 1960, p.348.
14, Stelos, Pi and Talmage, D.W.: J,Inf.Dis. 1004 12611967/.
' 15. Talmage; D.W., Freter, G,G., and Taliaferro, W.: J.Inf.Dis.
98:300 /1956/.
164 . Trnka; Z,\ and Franek, F.; Folia microbi01. 5: 374 /1960/4
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Formation of 19S and 7 $ Typo Viral Antibodies. The Role
\
and tfature of "Antibody Cofactor".
B Styk and L.H4na
Institute of Virology, Czechoslovak Academy of Sciences,
:Bratislava, Czechoslovakia
.In recent years data have been published on the- formation
of 19 S and 7 S type antibodies in response to various viruses
'A 7, 1. at al./. in our contribution, findings will be present-
ed mainly concerning the nature of antibodies to influenza
viruaes. A further difference in the character of anti-influenza
antibodies will be described in addition to the 19 S - 7 S type,
-' i.e. the change in their ability to be potentiated by the
?
so-called."antibody cofactor". We will show that wIsloadinG
results in the detection of 19 S,and especially of 7,S influenza
.,antibodies can be obtained if the additive effect of cofactor
is disregarded. Pomo characteristics of this serum component
will be therefore presented first.
We found /4, 5/ that the-titres'of speCific influenza anti-
haemagglutinins decreased after heating of antisera /56 ocim Mins.j,
if -a "non-avid" A2 influenza strain /i.e. a strain of low anti-
,
?
body.sensitivity, also designated 42- /of 2/ was used as antigen
in the haemagglutination Inhibition test /HI/. The decrease was
luch more marked in sera taken after first virus administrations
than in hyperimmune sera /Fig.1/. It was possible to restore
,
? .the titre of HIT antibodies to their original, or even to a
higher, level /Fig.1/ by adding normal unheated serum containing
no inhibitors to A2-virus.
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Styk ot aii - 2'
',ho fact that' the iCtivity Of heated .or stored iMmune
seraincreases.after the addition of normal unheated serum,'
is not a new' finding in.Virology/Gordon, Mueller, Moyer, Morgan,
Mhitman,'Leymaster and:Vard,:Ohaock, Casals and .others/. Our
experiments., however, 6ffered- evidence that, at least with
influenza viruses, the:pptentiating factor is not complement,
but an entirely different thermolabileserum component. We
Proposed. the name "seruM cofactor" or "antibody cofactor" for
:this component.
Several approacheS. wore used.to prove that the cofactor,is
not identical with Complement or its.components /el --C'4 /.
Clear evidence was obtained when comparing the titres of cofactor -
and C'eomponents in sera freed of complement by. binding on the '
antigen-antibody. complex. .Further, ?substances with anticomplemen-
tary activity, such as dextran sulphates, heparin, pelentan
and trypsin, or the removal-of the O'3 component of complement: by
zymozan treatment, de not substantially affect the cofactor
titre.'HThe presence of EDTA, which. blocks the activity of q'l
:component of complement, doeshot inhibit the cofactor-activity
.in human, mouse,: pig, piglet and horse sera.
With_some.animal.sera we iwore successful in separating the cc-
;:factor from the components of complements, using gel filtration.
- .
-Separation of guinea pig serum an Sephadpx G.7.200 is illustrated
Fig.2. Cofactor activity ocCurred mostly in the first peak
containing the macroglobulinS, -whereas the individual complement
'components were mainly present' in the 7 S peak. It must be added
that with many other sera the first C'componentwas.also detected
in the.macroglobulin peak. /C components wore'.detected using
-.reagents R.1, R2 R3 and R4, deficient in the- respective C'component
?
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011 paper electrophoresis, the cofactor from mouse serum
.. moved along with :.%globulins...On immunoelectrophoresis, the
cofactor aetivity of bovine serum wds bound to the !2-macro-
globulins. The macroglobulin character of cofactor from various
:Animal sera was also demonstrated by density gradient zonal
centrifugation /3/. In Fig.3 the macroglobulin nature of mouse,
bovine and pig cbfactor evident. However, the cofactor
from serum of newborn unsuckled piglets is of lower molecular
weight showing an s20, value of 3.0 to 4.0 S. The smallei,
molecular size of piglet cofactor was also confirmed by 'chromato-
,graphy of piglet' serum on Sephadex G-200 /Fig.4/..
Now some remarks on the 19 3 and 7 S influenza antibody
'formation. The character of .antibodies in rabbit serum taken
10 days after administration of non-avid A2-influenza virus
-Is illustrated in Fig.5. Both 19 S and 7 S antibodies can
be detected in unheated ?samples against. the homologous antigen.
The titres of these anti-haemagglutinins decrease after heat..
? ing /56?C/30 mins/ but can bo restored by adding cofactor in.
tho form of normal mouse serum, -.Using the A2+ virus as antigen
ip the HI rest, the 7 S globulins can also be detected in this
- antiserum .though in a low titre, which is rather surprising.
We cannot saji however, in what quantity 19 S anti-A2+ anti-
? bodios are present in this antiserum. The samples from the'
macroglobulin peak show quite high HI activity, but wo
found that the nonspecific r inhibitor /against A + strains/
is present in this peak after SephadeX chromatography of
normal era4;
The Sephadex separation of another rabbit antiserum is
illustrated In Fig,6. The serum.was taken 10 days after A2+ the
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Styk et -al. -
administration of inf14enza virus. The-fortation of 7 S anti-
- ,
\
bodies against the homologous antigen is obvious. The 19 S antibo.
dies aro alsO Probably present, but as in the previous case -
they cannot bo differentiated:from the non-specific
Both 19 S /in a small quantity/ and 7 S antibodies can be detected
against a non-avid /-/A 2.strain, but only after addition of the
' serum cofactor, the importance of which thus becomes clearly
? evident.
Extensive immunisation experiments on white mile revealed
,great variability as far as 19 S and.? 3 antibody response is
concerned. 'There were cases where 19 S antibody formation preceded
7that of 7 S antibody, as expected. However, in other experiments,
7 S antibody appeared simultaneously with, and in equal or even
higher amounts than 19 S'antibody. This ocured on the 3rd -:5th
day. Detoption of antibodies . or/their,titre depended on
?
, whether serum cofactor was present in the respective fractions.
'
?-
?This is especially true of 7 S antibody, because owing to the
macroglobulin character of cofactor, the lator was not present
in fractions from the 7 S globulin peak. A similar case is shown
in Fig.7 which illustrates the separation' of mouse serum taken
5 days after virus administration. Judging from result 6 which
:samples tested in saline, a small amount of 19 S and 7 S antibody
should be present. However'. if the same samples are tested with
cofactor added /1:06.. using normal mouse serum 1; 15 as diluent/0
iho detected amount of 7 S antibody increases considerably. This
is also true for virus neutralizing /VN/ antibodies. /VN acti.
vity was 'tested only infractions from:the top of the first and
?second protein peaks/. In many cases the activity of 7 S antibody
was much more enhanced by tho cofactor than that Of 19 S antibody.
,
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Styk et al. -5
' 04 sumMary0. we would like to point out that in addition to
the replacement of 19 S .by 7 ?S antibodies, a further change in
the character of, antibodies froni "early" and "late" _influenza- anti-
sera can be observed, i.e. their ability to be potentiated by the
."antibody cofactor": The former change /193 7 S/. is usually
_-.more rapid and does not coincide with the latter because anti- '
body which is and that which is not potentiated by the cofactor
can be both of the 7 S :type.
Serum'cofactor does not play any role in the activity of
antibody /either 19 S or 7 S/' to bacteriophage Cr) X 174. Our
results of 19 S and 7 S antibody formation were similar, to
those in experiments of Uhr and Finkelstein /7/. However, in
addition to the expected sensitivity and insensitivity of 19 S and
of 7-S antibody, respectively, to mercaptoethanol, we observed
the formation in rabbit of 7 S type .nti-phage antibody which
was sensitive to 2mercaptoethanol treatment.
References
1. Berlin, B.S.: Proc.Soc.exp.Biol.Med. 113:1013 /1963/.
2. Choppin,, P.W. and Tam, J.exp.Med. 112:895 /1960/.
?,
3. Styk, B,, liana, L. Frank,'Pt., Sokol, and Men5ik,
J.
Acta virologica 7: 25 /1963/,
Styk, B., Ko6i5kova, D? and B1a6kovi60 D.: Acta virologica
3 /Suppl./: 97 ./1959/.
Styk, B? RathovA, V., and B1agkovi6, D.:. Acta viroiogica
2:179 /1958/.
Svehag, S.E. and Mandel, B.: Virology 18:508 /1962/.
7. ? Uhr, J.W. and Finkelstein, Joexp.Med, 117:457 /1963/.
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%.9g!J WEAL UAL (MU
?
-; ?
rq
. ?
The'.0dcurend& and Significande.of Natural Antibodies
.; ?
,Boyden
? ,
.,.,Tohn-Curtin School of MOdical. Research, Australian National
?
University, 'Canberra, Australia
I.
The means by which the antibody-forming system discriminates
betwoen-material.to which it is naturally responsive and that to
- .
Which it is naturally unresponsive is unknown. The point is a
ver crucial crucial one in immunological theory /20, 19/ and know-.
ledge Of the mechanism Ohe. discrimination would todoubtebly
help towards an understanding,of.the whole proceSs'of antibody
.formation..
A 'similar problem faces those, who are interested in phago-
fluids t /6, 7/- A.difference between the .two problems is.that
iiecogr4tich. by the antibody-forming system seems only to apply to
-
dytosia, for the phagocytic system of multicellular animals also
possesbes a remarkable discriminative capacity. The resemblance
of these two'problems is :indeed close, for in each instance he
dells appear to "recongize" and respond positively to the same
-category of substances: in both tho antibody-forming system and
the phagocytic systemi.the response appears to be elicited by
::lacromolecular groups which are not normally present in the body
4,4
+For watt of A better term, the Word Itforeignil will be used to
denOtelthaterial, Whether autohhotonous or of extrinsic origin, which
?
is not normally present or exposed in the bodyfluids.
.1
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Boyden-2
vertebrates, whereas recognition of foreign matter is a
property of phagocytes throughout the metazoa.
The suggestion -has been put forward that the means by which
- phagocytes, invertebrates and invertebrates, identify foreign
matter is the same, or at least very similar, to that which
'operates in the immune response /6, 7/. It seems reasonable to
postulate that since a very efficient mechanism of discriminat-
ion between foreign and not-foreign matter was already present
in the invertebrate ancestors of the vertebrates, the same mecha-
nieM may have peen utilised during.evolution to form the basis
of discrimination by the cells responsible for the production Of
antibodies. It must be admitted that this concept is not com-
patible with the clonal selection theory /19/, according to which
immune discrimination is due to the presence in the bddy of a
vast number of genetically different cells, each responsive only
to one ora few foreign antigens, and to the absence of mutants.
which would be capable of responding to normally:exposed self-
components. Obviously such a mechanism could not account for
discrimination by phagocytes, which are mUltiponeential with
regard to the macromolecules which they appear to recognize
as foreign. -However, we feel that a more complete examination
of the probloffoftdiscrimination by phagocytes might help towards
an undurstanding of the first essential step in antibody formation-
,
thePrecognition of foreign antigens,
A number of theories of antibody formation require the
existence in,the body fluids, or on the surfaces of cells; of what
.may be termed "recognition factors" /24,-68, 39, 581 5/.
- According to these theories, recognition of! the antigen depends
on the presence. of special substances which combine, with foreign
,
determinant groups in the initial stage of the process of anti-
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Boyden -k 3
? body formation, serving initially to link or associate the
foreign molecule with certain cells. Only recognition factors
reactive with foreign or unfamiliar determinant groups are
present.
? .These.introductery remarks provide the background to
- our interest in "natural antibodie". The question we ask is'
whether' "recognition factor's" can be det9cted in non-immunised"
'animals, and whether natural antibodies, or certain types of
? natural antibodies, might be the postulated recognition face ors.
? At. the present state of our knowledge the best we can do 'is to
Consider whether the information available about natural anti-
bodies is compatible with this notion, and we will do so bearing
in mind the concept tat recognition by phagocytes and by the
'antibody-forming system may be due to essentially the same
mechanism.
At this point sOmothing must be said about the term "natural
antibody", in order to avoid, as far as possible, unnecessary seman-
tio debate. Difficulties arise if we follow rigidly any of the
usual definitions of antibody. Some definitions, for example,
insist that an antibody must be a globulin. But if we find sub.
stances in the blood of, for example, an invertebrate with the
same sort of biological activity and function as the gamma-
globulin antibodies of vertebrates, but with different electro-
,p4orotic mobility - are we then precluded from calling them
antibodies? The requirement that antibody should react specifically
with antigen raises difficulties which will be mentioned below.
Biological activity does not provide a good basis- for a definition.
Many virologists use specific neutralisation/of virus infectivity
as a test for antibodies; nevertheless gamma-globulins are some-
-times detectable in sera of immunised animals which react spocifi-
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Boyden - 4
. .
..callyWith-virUd but which enhance its infectivity /30/; Nor can
. .
phenomena such agglutination, aomplement fixation, opsonisation
etc; be used as a basis for definition,' for ,not all antibodies
give positive reactions' in these tests even when the antigen is
suitable. ,
Further discussion on this point is not worthwhile. In this,
paperp.the term' natural antibrdy will be. used in ayery .broad
sense for substanceS /probably always protein/ which are present
' in the body fluids of.normal,animals and which can be shown to
combine with foreign matter but not .with normally exposed "self-
,coMPononts"; it' is assumed that.natural antibodieg-have some
role to play in keeping the tissues free of extraneous and
unwanted matter although this function is not, of course, always
demonStrable.
Natural Serum Antibodies
:Occurrence
' The fact:sthat norMal'.serum fr.= many, different spebies of
.vertebrates contains s,ubstances with physico-,chemical and bio-
logical properties.similarAo those- of the antibodies which
'appear, usually to much higher levels following immunisation
-ha's boon known since the 'end of the last centrury. The referen-
ces:in. the ,literature te such:natural antibodies are innumbrable;
. it is almost impossible. to pick Up?a number of a'current immune-
:logical journal Which does nOt.contain some reference, direct or
indirect,, to natural antibodies. The subject has' frequently been
discussed in textbooks and review ar:ticles /88; 73, 47, 500' 71/.
: Natural antibodies :have been described in vertebrate sera
.reaCtiVe in one test oranother with very many different kinds of
bacteria /see reviews mentioned above/, with bacteriophage /40, 80/0
. .
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? '?
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.-?
'Boyden - 5 .
? , /?
, . i ? . ? ,
zymosan /61/?. starch particles /620.81/, protozoa /72/, fungi
' ? .
, ? ,
,/15/, mdtazoal parasiteS /70/, heterologous and isologous ery-
tthrOcytes./56; 31,.2,.47/ and mammalian tis;ue:cells /48,, 77/,
Sera of normal animals have also been shownto contain anti-
?
bodiasagainst components ofthe host not normally exposed in the
_cirpulgtion,'inCluding spermatozoa '/53/ and extracts of various
.tiasues /43, 9/..- z.
?
Natural.antibodies.appear to have been demonstrated in sera
of all vertebrate speCies exmine,d, including reptiles and fishes
'/see 72/. Some authorslhave attempted to rank different,species
according to the levels,' of .natural antibodies in their' sera /18,
28/, but the ubefulneea.ok'thia procedure is 'doubtful, espe-
Ciallysincevthe authors differ in the order'in which they place
the different species.
There are also many repots of substances, in the body fluids
,
of invertebrates with in vitro biologica: activities similar to
? those of antibodies in: mammala. Bactericidins, haemagglutinins for
.varioua animal cells etc. have, been described /911, 35, 21, 84/,
thus clear that' normal sera'from. all'vertebrate-ariiffials
and .many invertebrates Posseas antibodylike 'activity detectable
,by one procedure .or another against a vast array of antigens. In
cdnnection with the preaent topic the. important:queation arises
:whether, or not natural antibodies detectable in the sera of vert'e-
brates have been produced in response' to 'an antigenic stimulus,
sperhaps:from a foreign Macromolecule .which shares determinant
:
groups With the test antigen., lially= authors /e.g.87/ believe that
.this tobe,the case in the vertebrates; and there can.be little .,
doubt that soMe of the, antibody activity of-normal serum is due to
'this causes There is plenty of 'evidence that many antigenic ,deter-
/
mingnt groups are shared' among microorganisms and animal tissues;
? ;
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?
.?
-Boyden - 6
the?b4Ssman Antigen is rdbost *.ell.knOwn example /26, 16, 4/.
?The feet that titreSoftany naltral antibodies increase with
age iscompatible with this view /22, 88/.:
? 1.
If all naftUral antibody Activity were the consequence Of
previous antigenic stimqlation, then the idea that the postulated
recognition factors are detectable aS natural antibodies would
? obvieuSly not be tenable. However, although stiMulation by cross-
reacting antigens is.respensible for some of the antibody acti-
vity of normal serum; particularlYswhen high titres are demonstrab-
le, dogmatic statements 'that all antibodies detectable in normal -
serum are the consequence of antigenic stimulation are not
justified at the present tiMe., Some authors, in fact,, take the
view that both the level and the specificity of- natural anti- .
-bodies are genetically determined /33, 42/1- and there are several
;-1
studies indicating that genetic !actors control the level in tlie ?
serum' of certain natural antibodies against heterologous antigens'
/52, 74-1 75, 44, 36/. It is well know, Of course, that the
production of certain' isoantibodies is under .genetic control.
The question.can be put another way, are natural antibodieS
detectable against all the avetigens against which. the host is
capable-of responding immunologically? An affirmative answer 'to
this question would be strong Ruppert for the recognition factor'.
.'hjpothesis, but unfortunately, available evidence does not justify -
ra definite answer one may or the' other. The problem does not seem
to have been attacked systematically, perhaps because the task would
'seem to beHtoo stupendous. However, a. , si . ngle Clear demonstration '
N
.that "natural" antibodies.do not exist against any Single antigen;
which a given'animal.is,immunolegically responsive would be
. .
dufficient evidence'to eliminate the hypothesis that natural anti-
' ? ?
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Boyden - 7
bodies are.the reeognition.factors for acquired immunity. Even
/
in such a case,, one could always argue that the test used was
not sufficiently sensitive. Certainly a negative result in one
of the classical techniques, depending on agglutination, passive
haemagglutination, bacterial activity and so forth would not
warrant the conclusion that natural antibodies are not present.
/t is Sometites stated, for instance, that normal guineapig.
\
.serum,contains no natural antibodies reactive with sheep erythro-
cytes /0 g. 54/. The resuls given in Table I show this statement
to be. iricorrect. It is true that normal guineapig serum does-not
amlutinate sheep cells, although it has fairly high agglutinat-
ion titr4s against pessum and rabbit )red cells. However, if sheep
cells, after e7posure to dilUtiens_of normal guineapig serum,
. are washed and resuspended in rabbit anti-guineapig serum, they
agglutinate to a titre of about 0560 of the guineapig serum,
The factor in the guineapig ..serum Whiah renders the sheep cells
agglutinable by rabbit antiyguineapig serum is speCific, in that
absorption of the serum .with'sheep cells completely removes it,
but leaves the titre against rabbit and possum red cells un-
changed,
.Another source of,error-is the possibility that the test
.mixture may contain substances which interfere with the.com-
.bination of an antibody with its antigen or with some observe-
able effect of this reaction. It. has been found that certain anti-
'bacterial antibodies interfere with the bactericidal activity of
other antibodies reactive with the same organism /1/. Thus, nega-
tive, results of a test'which depends on some s6conaary manifest-
ation of antibody-antigen interaction Certainly cannot be taken
as-evidence of .the absence of antibody.
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.Boyden,- 8
. ? ., .
Although an answer o the que'btion whether natural anti-
,
bodies to all potential antidens are present in vertebrate :sera
is not ye.(permissiblwe cannot but bo impressed by the fre7
clueney with' which naturai.antibbdies are found when we. look for
theM...Jt is relevant here to recall that it is a common corn-
,
.:.
plaint of Very sensitive serologicaI tests that "nen-specific"
,
reactions are detained With normal .sera.
,) ? ' ?
'Heat stability .
It is?sometiMes stated .that .natural antibedi0i.in mammals
are heat labile /56?C/ whereas "immune" antibodies are stable',at
this temperature /73/ ,ipweNier, the literature on this Point
extremely confusing, Ancl it would not .be worthwhile to deal with,
the question fully here. Some of the contradictions may be due to
the,fact,that heat treatment ,has been.carried ott at different
?hydrogen-ion coneentratiopsor at different dilutions, factors.
,khich can.influenee the heat lability of proteins /73; 55/, There
are.many other factors which may contribUte to the.cenfusion, in-
;
, eluding the fact, often forgotten, that heating does not merely
. -
. . . .
'destrey the chetical actiVities of certain labile substances; it.
also results, in the production of new activities which may inter-'
- .
fere an some way with serological reactions /ill.- One view that
? ? .
? 1,
'has been. expressed is that, among .the bactericidal substances in
normal Serum, those active against gram-negative bacteria are re-
.latiVely heat stable and ,require complement for their activity, ,
while those active against .grain-ppsitiv6 bacteria are relatively'
t.
less resistant to heat and ,do mot require complement /e.g.'stable
. .
at 57.59C0; but labile?at'6000 /55/1; but there are many contra-.
dictoryreports.on this point /23, 81, 32, 25/.. The significance
of such 7differences'is not Clear, but -the work of. Kleckovski /45/,
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Boyden, - 9
and of Bawden and: Eleckovski /3/ suggests that apparent dif.L
? , ? .
'forences ixiheatstabiliitY May-in fact be, duo not to basid
'differences in the antibodies themselves, but rather to diffePen-
'ces in, the antigens4
It would not be Useful, here; to attempt to compare in much
the,resultS of-different workers using so Many different
'
,-techncities to -study different readtions of different antigen
with normal 'sera from different specJieS.,
Specificity
The ge,moral staterio\nt iS often made that-true natural'anti7
todies are less specific tihaii "immune antibodies, ana_aro lose
absorbed out of serumIBB/: On the other hand, there are
many ./;eports:of highlY,Specific antibodies in noinal serum .
,00,.59:, 1, 81,,36,'31 17/. Twd factors need to betaken into
;account in this connection. First,- the difficulty in completely
absorbing antibody activity Ott of normal serum as compared with
immune serum does' not mean 'that there is any fundamental physico-
chemical difference between the antibodies in the two cases;
,it.maYmean.only that the proportion of antibodies with high
avidity for the antigen,i Hgreater'in the serum of an Immunised
'animal than in noinril serum, fact which May be_anticipated-from
N
any selection theory of antibody.fgrmatiOn /76/i Second when
, ? .% . i
?
. . - ./- .
-comparing antibody activity against different antigens, the *Ion-
,
--stratiOn that-the antibodies against one antigen.are Mare ?specific
than those against another dees not neeessarily.mean, as is often
y implied, that one is. dealing with two different Xinds 0
of-antibody. In many such eases it is just as likely that it is
the
the antigens.which differ, one being more .common in nature than ishe
Other. Thus,' relative' non-specificity can be anticipated in anti-
? ?
-
$
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!
active .Wilith aniniAl antigens, each specific for an antigen characte-
.
. .
ristic ,of whole aroups of aniMals. There are some difficulties
. Boyden 10
. ?
bodies' reactive With polysaccharides, since 'there is, much more
sharing. of .determinant groups among- the polysaccharides in dif-
ferent species of plants and :animals than amOng. the protein's. ,
. Some recent experiments :in our laboratory :have Confirmed the
i ?
suspicion that there is an apparent trend7towards A decrease in
? Nt
specificity of .natural.untibodies . as ye go backwards in the
evOlutipnary scale.
Tyler and Metz /84., 83/- have suggested that the body fluids
of certain invertebrates contain relatively few' antibodies re-
in accepting .this. view and -further' work needs to be done on the
:subject.
:
Recognition lby phagocYtes
1
Ii has been known for 70 years that phagotytosis by leuco-
H
' cytesA of. mamnials is promoted bY components of 'normal serum which
, were Called ".opsonins" by Wright and Douglas /89/. Opsonins became.
-
Yi
adsorbed.onto the surfaces of :foreign particles which are thereby .
rendere4 attractive te the phagocytic cells: 'Much Work since ,that
:time .ha S shown that in Most cases two: factors in normal serum are
involved in this activity, the one heat stable /56?C/ and' specific,
'the 'other heat labile and non=specific.' The latter factor is often
,conSidered to be complement; the former is natural . antibody. There
however, no general agreement in this field /see 11/; it has
.retentlY been claimed, :for example, that the opsonic activitY,t1f.
:normal rabbit serum against certain' bacteria is due 'to' a single
, .
non spepific heat labile substance, which is not complement /32/. .
While there is still, some corfusion as to the exact nature of
' all the "opsonic factors in normal serum, there is nO doubt that
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Boyden - 11
'serum factors which combine With the surfaces of foreign par-
, /
? :tidies are of immense importance in phagocytic recognition. In most
cases:nor interaction oecurs'between phagocytes and partieles if
the-latter are not coated'with host gamma-globulin.
There is. evidence that natural antibodies also play an
essential role as recognition factors not only in the actual
process of phagocytosis, but also in the'first phase'of
.leucocYtic response to foreigh particles - chemotaxis. For
instanbe, the chemotactic effect of cellulose ?and foreign
-red cells on rabbit polymorphs depends on the adsorption onto
the surface of the particles of serum components. The results
summarised in Table 3 indicate that cellulose is not chemo-
. tactic for'polymorphs in Medium adsorbed with cellulose, in the
Cold, although.sheep red cells are chemotactic in thislmedium.
? Conversely, in medium absorbed with sheep red cells, Cellulose
elicits 's. response, while sheep red cells do not /not shown
in the Table/. As in the case of complexes between soluble anti-
gens and their antibodies, the chemotactic response seems to
_
"depend on the interaction of antigen-antibody complexes with
non-specific heat labile components of serum,
Information bearing on the mechanism of recognition by the
'phagocytes of invertebrates is almost entirely lacking. However,
in some recent, experiments in vitro phagoCytes of the snail
/Helix aspersa/ were found to phagocytose formalinised sheep
red cells in balanced salt solution only if the latter had 'been
pretreated with snail serum /North and Boyden, unpublished/.
Earlier Ruediger and Davis /67/ demonstrated "opsonins" in the
blood of a number of invertebrates active against a' number of bacte.'
ria. Unfortunately these authors were studying the uptake of the
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Boyden - 12
'treated bacteria by Mammalian phagocytes ih-Medium containing
mammalian serum. ThUs,while; they 'certainly showed that the.
,
:insect blood modified the surface of the bacteria, they did not
eliminate the possibility that the opsonic.activity migh have
been due to the adsorption of mammalian serum components onto
the modified bacteria,Jperhaps by reacting directly with acl.
,-?sorbed insect proteins.,
;Cell-bound recognition factors
It has been suggested that the specific host substance 'which
;combine& with the antigen in the.intial phase of the immune
response is present ,the surface of the potential antibody-
'producing ,dells /24, 58, 5-, .6, 7/. In this connection it is
"pertinent to mention. that there exits in rabbits a kind of
antibody which, while' present in the serum,. also has a strong
:affinitylfor spleence1ls /13/. 'The cells, when coated with this
antibody, are capable of speeifically adsorbing antigen. In.a .
.-recemt-study a similar "cytophilic* antibody has been demonstrated
:in guineapigs Itr:lunisod with sheep red cells mixed with complete
.Preund's adjuvant, and in these experiments ,its presence was
found 'to be correlated with the existence of a state. of delayed-
/ ,
type-hypersensitivity./8,. 10/. Guineapig macrophages which have
- /
been treated with. this antibody have a strong affinity for sheep
.red cells, and cytophilic antibody can therefore be said to play.
role in the reco,Lnition'hy?these cells of the foreign particles.
There 'is also some evidene suggesting that an antibody'of this
' type,,,present in nortalguineapigsi. is important in the recognit-
ion by,macrophages Of 'effeteautochtonous erythrocytes /85, 86/.
.,The,signifieance'of natural autoantibodies has benn discussed
elsewhere /Boyden, 1964a; Grabar, 1958/.
OR 'UTICA
SE 'of.,
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Boyden..: 13
Furtherwork is pecesSary to show whether cytophilic or cell-
.
bound antibodies are a1way6 present .in small amount in normal
animals; but on the face of it, it would seem that. natural*
'cytophilic antibodies are good candidates for the toles of
recognition-factors in' antibody formation. *
Comment
In spite of the.large emnOnt of work dOne on natural anti-
bodies, it is not poSsible at present- to *draw any definite.
condiusions as to their nature and. origin. We may'safely.state,
'however, that the, body fluids of both vertebrates and inverte-
brate w have a very broad range of reactivity towards different
foreign antigens, and that the natural antibodies responsible
-for this reactivity are the mtan, Means by which the phagocytes
of vertebrates and possibly those of invertebrates discriminate
'between foreign and non-foreign matter. The natural antibodies
have; been separated into groups by various workers on the basis
of differences in heat Stability, range of specificity, electro-
phoretie.mobility /66/, weight /34., 46, 27, 82/0 cytophilic
properties and so forth. While the. significance of- the various ;
differences is by no means clear, at present, it is not impossible
that one of the groups represent the postulated "recognition
, factors" of the immune response.
:Of possible relevance in this connection is the i-epOrt. that
the capacity of 3 week old baby pigs: to produce antibodies against
;. certain antigens* is decreased if they are deprived or maternal
coloStrum.--Antibody -production in the colostrumdeprived.pigs
\
is said to. be' stimulatedby mixing\small amourits of' horse anti-
body with the.antigen:at imMunisation /69/-. It has also been shown
that antibody production in mice /78/ and in rabbits./51/ is
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Boyden --14
? greater if.antigen antibody complexeS are injected /in antigen
excess/ than if the antigen is 'injected alone. Perhaps experi-
mental use could .be made off the intereting observatiOns that
the .general. level Of natural antibodies, /unrelated Serelogically
tq the .stimulus/ increases Markedly in various illnesses /37/
and also following the Anjeotion of certain bacterial 'oducts
,?
/50, 64, 650' 313,57/.
The time As now ripe for a new onslaught on natural anti-
bodies, using hew.physico-Chemical techniques, and prefer-
ably very sensitive serological methods which detect antibody-
. antigen 'combination directly and whiCh do .not depend-on some
:unreliable manifestations of the interaction.
References
1. Adler, F.L.: J.Immunol. 70: 69 and 79 /1953/.
2.;. Bailey, C.E.: Am.J.Hyg. 3: 370 /1923/.
3. Bawden, F,.C. and Kleckowuki A.: Brit.J.exp.Path. 23:
178 /1942/.
? Boyd, W.C.: Fundamentals of Immunology, Wiley /Interscience/,
New York 1956.
. Boyden, S.V.: Nature 185: 724 /1960/.
. Boyden, S.V.; J.Theoret.Biol. 3: 123 /1962/.
7. Boyden, S.V.:,Internat.Rev.Exp.Path. 2: 311 /1963a7.
8. Boyden, S.V.':'Cell.e.Bound Antibodies, Wistar Institute Press,
Philadelphia, U.S.A., p'.7 196310,
9. Boyden, S.V.: Nature 201: 200 /1964a/.
10. Boyden, S.V.: Immunology 7: /1964b/. In press.
11. Boyden, S.V.: Ciba Foundation Symposium on "COmplement",
G.E.T. Churchill, London, 1964c. In press.
. ? ,
S
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' Boyden - 15
Boyden, s.y, ar*1 sor1041,..E.2 Immunoiogi.3; 272 /1961/.
?
13, Boyden, S.V, and Sorkin, E: Immunology 4: 244 /1962/.
14.: 'Boyden, Roberts, J: and Tait, N.: Unpublished
results, 1964.
.15,i Brody, J.I. and,Finch, 6.c.: Blood 15:-'830 /1960/6.
16., Buehbinder, L.:A.M.A. Areh.Patho1,1:19: 841 /1035/.
Buliech, W.B. and Western, G.T,1 Proe.Roy.Soc.B., 77:
531 /1906/..
18. BUrgi, E.: Areh;gyg. 62: 23911907/.
\
19.i 'Burnet; F.M.: The Clonal Selection. Theory of..Aequired
Immunity, Cambridge University Press, London/New York .
1959. ?
20. Burnet, F.M. and Penner, F.: The Production of Antibodies,
Melbourne, Australia.1949.
21.j Chorine, V.: C.R. Soe,Biol. 97:1395./1927/. ,
.22.'] Cohen, .3. and Newton,.WtL.: 3.ImmunoliL90: 358/1963/.
Dunlop, EM,. JoPath.Bact. 31: 769 /1928/.
25.
26.
Ehrlich, P.: Proe.Roy,Soc.B.
66; 424 /1900/.
Ekstedt, R.D.: J.Bact, 72: 157 /1956/.
Forssman, J.: Bioehem.Z, 37: 78 /1911/.
.27. 'Gabriel, M.J. .and Rosen, F.F.: J.Exp.Med. 118: 619 /1963/.
-- 28. Gibson, H.J. J.Hyg. /Camb./ 3,37./1930/.
- 29. Grabar, P.1,proc,Sixth Intern.Cong.Haematol.,, Grune and ,?tra-
tton, New York, -033, 1958.
Hawkes, R.A.: Submitted for publication, 1964.
?
31. Hektoen, L.: J.Infeet.Dis. 5: 249 /1908/4
32. Hirsch, J. and Strauss, B,: J?Immunol, 92: 14511964/.
33. Hirszfeld, L.; Ergebn.Hyg.Bakt, 8: 367 /1926/, .
34. ..liogman, C.F. and.Ki113:nder, J,: Acta Path.Microbiol.Scand.
-;55057 /1962/t
.1 ?
?
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Boyden - 16
35.; Huff Physiol.Rev. 20: 68 /1940/.
36. Irwin, M.R. and Bell; J.Inf.Dis., 57:. 74 /1935/.
374. Jacox, R.F.:?J.Exp.Med. 92:101 /1950/.
38. Jenkin, C.R. and Palmer, D.L.: ?13-..4cp.Med. 112: 419 /1960/.
39 Jerne, N.k.: Proc.?Natl.Acad.Sci. U.S; 41: 849 /1955/.
40.; Jerne, N.K.: J.Immunol.,76: 209 /1956/.
41. Jordan, 2.0.: J.Inf.Dis. 61: 79 /1937/.
42. Kershaw, 13.B.*: Yale $T.Biol. Med. 21: 463 /1949/.
-43.. Kidd, J.G. and Friedewald, W.F.: J.Exp.Med'. 76: 543 and
557 /1942/.
44. Kirshbom, I. and Hoecker, G. Nature 200: 687 /1963/.
45. Kleckowski, A.: Brit.J.Exp.Path. 22: 19.2/1941/.
46. Kunkel, H.G.: The Plasma Proteins, Academic Press,' New
York/London, 1960.
47. Landsteiner, K.: The Specificity of Serological Reactions,
revised edition, Harvard University Prpss, Cambridge, Mass.
U. S.A. 1945.
48. Landy, M., Michael, J.G., Trapani, R.J., Achinstein, B.,
Woods, M.W. and Shear, MJ.: Cancer Res. 20:1279 /1960/.
49. Ledingham, J.C.G.: A System of Bacteriology, H.M. Stationary
Office, London, 6: $1 /1031/.
50. Ledingham, J.C.G. and Bulloch, W.: Aberdeen University
Studies, Aberdeen, ScOtlandi, 1906,
51, Leskowitz,.S._: Fed.Proc., 17: 522 /1958/.
52. Linscott, W.D.: J.Immunol. 86: 480 /1961/.
53. ?Lumsden, ?T. 7*and Kohn-Speyer A.C.: J.Path.Bact.. 32: 181
/1929/.
54.- ' Maal/e; O.: On the Relation between Alexin and Opsonin,
'Munksgaard, Copenhagen., 1946.
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.-Boyden - 17
65.. !Maokie,. T.J. andFinkOlstein, -M.H.:J.HYg. 32: 1 /1932/.
!,56. 1\fidIkoff.,? GiM.1..Deusche Med.Wochenschr,.. 26: S-229 /1460/. .
A 1
57.-1 Michael, ?J.G. J.L. and Landy, 'Nature .191i 296
.
4
/19 '61/
58.- !Monod, J.: Cellular arid Humoral Aspects of the Hypersensitive
'J States, Hoeber-Harper HNow. York; 628, 1959.
59. .j/Junoz
J. 'and Hoiford, F.E.: J.Immunol. .63: 51 /1949/. ,
60,Mugehel, Proc.Soc.Exp.Biol.Med. 103: 632 /1960/.
61..
Nelson,
R.A.: J.Exp,Me4. 108: 515 /1958/.
62.
Nelson,
R.A. and Lebrun, Z.: J.Hyg../Camb./ 54: -8 /1956/.
63.
*Perkins,
E.H. and Leonard, M.R.: J.Immunol.
90:
228
/1963/.
64. 'Rowley, D.: Brit.J.Exp.Path, 37: 223 /1956/.
65. !Rowley, D.: J.Exp.Med. 111: 137 /1960/,
66. Rowley, D. and Jenkin, C.R.: Immunol. 5: 557/1962/.'
67. Ruediger, G.F. and Davis, D.J.: J.Infeet.Dis. 4: 333 /1907/.
68. Sahli, A.: Schweiz.med.Wochenseter.: 1:1129 /1920/.
69. ?Segre, D.,and-Kaeberle, M.: J.Immunol. 89: 782 /1962/.
70.
Sewell M.M.H,1 Immunology 6: 453 /1963/.
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;
74.? Stern,..K. and DaVidsohn, 'I.: J.Immunol. 72: 209 /1954/,
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? Terres, G. and Wohn, J,Immunol. 86: 361 /1961/, ?
79... 'Tiniourian, H and:Dobson, 150:. 27 /1962/.
80.' .Toussaint, A,j: and Musehe1, J.ImMUnol 89: 27 /1962
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? .
Boyden 18
81, H Tiirk, : Brit ?J?Exp.Path. 40:578- /19.59/.
82. Turner,. K.J. and 'Rowley, D,: Aust.J.Exp.Biol.MediSic,.
.41: 595 /1963/.
83. ; Tyler, Bull. 90: 213 /1946/. '
1
84. Tyler, A. and Metz, .C..G.: J.E)ip.Zool-. 100: 387 /1945/.,
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86. Vaughan.. Rs and Boyden S.V.: Immunology 7 118 /1964/.
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'?Wilson, G.S. aridliales, A.A.: Tapley and Wili3oh 's Principles
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89s Wright, A&E. and,Douglas, S4R.: Proc.Roy.Socy. /London/,
? B. 73: 136 /1904/.
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?
?
91. Zernoff, V.: 116: 30411934/. ,
?
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1
, I' 7 g ,?
it Declassified in Part - Sanitized Copy Approved for Release 2014/01/15: CIA-RDP80-00247A004200090001-4
erum 'dilutions
Boden - 19
? ! ?
? ? ?
;1/ / . 1/ 1 1/
!10 20 :40!
. .
V 1/
80 100
1/
00
1/ 1/
640 1280
1/ ? 1/ :1/
2560,5120 10240
Saline
Control
1 .
Directi
itest 1
,
!Sheep'.
!red ? ! ,
1.
!cells .1
' ? !
'Rabbit i
red .
cells j
?1
Possue i
red --+
cells
Anti-?? ?
globulin
test
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red !..0
cells;:
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cells,.
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'
Tvv.of6,1.d dilutions of normal guineapig serum /heated 56?30 /
were made in 0.4c c. quantities ?of saline.? 0.1 cc. pf a 1% sus-
pension of red cells were added to each tube. The tubes were
left overnight on the bench and the test. was read /Direct test/.
:The cells were then waShed once in Saline and resuiPended
ac. 1/50 rabbit anti-guineapig serum and a second reading made
after. 4 hours /Anti-globulin; test/.
?
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Iliad , ,
7 Declassified in Part - Sanitized Copy Approved for Release 2014/01/15: CIA-RDP80-00247A004200090001-4
p.--
-
;
? - ?
?
? ? 1
' Table
. ?
)
Effect of absorption of sertird.with
Boyden - 20
Migrating cells
Medium;
Absorbe.d Medium
1
0
Medium and Cellulose ? . 143
Absorbed. Medium )- Cellulose . 16
/ ?
Medium + SheeP Cells 132
Absorbed Medium + Sheep. Red, Cells t ? 155
,
Medium, consisting of \ 20% Normal Rabbit 'Serum, .was
,
'absorbed with cellulose at 00C /100 mg. cellulose per 01 cc
,
'serum/ The figures refer to '?the number of polyinorphs /per
field, 400 x magnification/ which have migrated through
a? Millipore Filter Membrane /ave.pore 'size 3 micron/ towards
the test mixture -./see 6/.
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NUM
r- Declassified in Part - Sanitized Copy Approved for Release 2014/01/15 : CIA-RDP80-00247A004200090001-4
e.r11 -
,r7Firm!
"CS
Carbohydrates Heterogeneity in li,abbit 7 S Gamma Globulin
E.M. Press
Depertment of Immunology, St. Mary's Hospital Medical
School, London, Great ,Britain
Gamma-Globulins from several species have been shown
to contain a small ;amount of carbohydrate which is covalent-
ly bound to the protein. 7 S'Y=globulins contain about 2 -
2,5% of carbohydrate consisting of mannose, galactose,
hexosamine sialic acid, and fucose, thyugh the content
aof' fucose is less than a mole/mole for rabbit Y-globulin.
Although the biological function of the carbohydrate is
not known, it is an essential feature of the structure, and
chemical studies have been carried out to determine whether
there are one or more oligosaccharide units on each molecule
of Y-globulin.
Emil Smith and co-workers'isolated glycopeptides from
papain digests of heat denatured. Yr-globulin and concluded',
from amino acid sequence studies on the glycopeptides, That
human, bovine, and rabbit Y-globulin contained only one
oligosaccharide unit /13,8,9/. However, Porter /11/ found
that when native rabbit i-globulin was split into three pieces
by digestion with papain, about three-quarters of the hexo-
samine ws present on one pf the pieces, III, and the other
quarter was associated with piece I. Also, the ratio of
hexose to hexosamine of IlIwas.bout 1,0, whereas for piece
I it was 1.35 /4/. This suggested that there were two
oligosaccharide units on the rabbit Y-globulin molecule and
, that they were of differing composition. Stmilar results were
reported by Franklin /6/ for human-globulin.ft
_
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Press - 2
It was shown by Edelman /2/, by Edelmam-and'Poulik
/3/, and by Frank /1961/ that when r-ilobulins are re-. ?
duced in 6M urea, their molecular weight falls from about
150,000 to 50,000, indicating that Yglebulin of all
species studied contclined"several peptide chains joined
together by disulphide bonds. As a result of further studies
on the products of reduction and their relationship to the
products of papain digestion, the four-chain structure
shown in Fig. 1 was postulated /12,4/. The two A chains have
' molecular weights of 50,000 and the B chains 20,0O0/10/.
Analyses of the isolated chains revealed that 95% df the
carbohydrate was covalently linked to the A chains /4/ and,
since there are two A chains. per molecule, it iS assumed that
there is one oli-gosaccharide unit on each A chain or two
? to each molecule of Y-globulin. The carbohydrate composition
? of rabbit Y'=?globulin and the isolated chains is given in
Table 1.
The value of four hexosamine residues A chain is in
agreement with the analyses of the glycopeptides isolated
by Nolan and Smith /8/ from a papain digest of heat denatur-
ed rabbit r-globulin /Table 2/. They also obtained, in small
? yieldp-a glycopeptide containing only aspartic acid and
carbohydrate and, by digesting the other?glycopeptides with
leucine amino peptidease and also by the Edman degradation
technique, they concluded that the carbohydrate was linked
to an aspartic acid residue which was in the C-terminal posi-
tion in these glycopeptides. We have digested fully reduced
rabbit Yr-globulin with pronase and separated the glyco-
peptides into two fractions which account for the whole of
the carbohydrate of the v:-globulin. Analyses of the two
glycopeptide fractions /Table show that there are four
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,
?
Press - 3
hexosamine residues/mole,aspartic'acid. Fraction 1 islorobab-
ly pure, and,carboxypeptidase A released free serine and
threonine but no aspartic acid, which supports the conclus-
ion of Nolan and Smith that the carbohydrate is linked to
,the aspartic acid residue. The hexose-hexosamine and sialic
acid/hexosamine ratios of the two fractions differ /Table 3/
but fraction 1 accounts for only 16% of the total he- .
xosamine. However, it is apparent that there is some hetero-
geneity in carbohydrate compositioh and, since the relative
yields of these two glycopeptide fractions is the same for
both fast and slow mobility T=-globulins, the heterogeneityl
is unlikely to be due to contamination with/other serum
components. Glycopeptide fraction 2 is presumably a mixture,
but both fractions could be part of the same amino acid'
sequence. On the other hand, comparison with the glyco-
peptides isolated by Nolan and Smith /8/ /Table 2/ shows
striking differences in amino acid composition, in particular,
the absence of phenylalanine from the pronase_glycopeptides.
From sequence studiei on their three major glycopeptides,
Nolan and Smith .deduced that they were all part of a single?
amino acid sequence
Glu - Glu /NH2/ - Glu /NH2/ - Phe - App
Carbohydrate
the aspartic acid being C-terminal and linked to carbohydrate.
From this they concluded that there was only one oligo-
-
saccharide unit attached to 1r=g1obu1in. As aal:.glycopeptides
contained no phenylalanine /Table 3/and in Noland and Smith
glycopeptides there was no serine or threonine /though g]zrco-
peptide 4 did Contain traces Of these nmino acids/ we assume
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-
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Press -4
that in the glycopeptides obtained by pronase digestion the
aspartic acid residue is N-terminal, and the sequence around
the carbohydrate deduced from our work and that of Nol'An and
Smith is: '
Pronpse Perin
,1 ?
Glu Glu /NH2 - Glu /NH2 - Phe - Thr/ /ProlGlu,
Lys/
Carbbgydrate
?;Presumably the pronase splits the peptide bond between phenyl-
alanine and aspertic acid, and papain cleaves the bond
involving the alpha carboxyl group of aspartic acid.
Although 95% of the carbohydrate or rabbit y=globulin
? is attached to the A chains, it was found, as stated above,
that carbohydrate,was associated with two of the three pieces
resulting from papain digestion of native rabbit y-globulin,
three-quarters with piece III, and one quarter with piece
I. As can be seen, from Fig.. 1, piece I consists of the B
chain and part of the A chain /A piece/ and these can be
isolated by reduction of piece I and fractionation on
a Sephadex G-75 column in N-propionic acid. When A piece and
B were analysed for carbohydrate, only trace amounts were
found. It was then discovered that the carbohydrate associat-
ed with piece I was not covalently linked to it but could
be dissociated from it, without prior reduction, at low pH
and it has been isolated as a glycopeptide fraotion by running
papAin piece I down a Sephadex G-75 column with N-propionio
acid /Fig. 2/. The yield of the glycopeptide fraction varies,
'or different preparations of r-glotu.lin-, from 20 - 30% of
the hexosamine content of the I-globulin, but there is no
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?