(SANITIZED)UNCLASSIFIED SOVIET AND CZECH PAPERS ON MOLECULAR AND CELLULAR BIOLOGY(SANITIZED)

• Document Type: CREST
• Collection: General CIA Records
• Document Number (FOIA) /ESDN (CREST): CIA-RDP80-00247A002800180001-0
• Release Decision: RIPPUB
• Original Classification: C
• Document Page Count: 102
• Document Creation Date: December 23, 2016
• Document Release Date: March 5, 2014
• Sequence Number: 1
• Publication Date: September 16, 1964
• Content Type: REPORT

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S.
A
Formation of Specific 7 S and Macroglobulin Typo, Antod100
in Chickens
Ieftiha
. Institute of Microbiology, Czechoslovak Acadamy'of Sciences,
,Prague, Czechoslovakia
The heterogeneity of antibodies has noW been demonstrated ?
- in, different characteristics of the antibody molecule; i-,e? in
. ? ? .
'serdlogical properties, physieo-cheMicU chiac-ter and ,in the:
antigenic structure /6/, Considerable data .have been Collected
oft the' formation of different types pf antibOdies4, For. ?oxariplo.i
.?
the immunization" of rabbits with foreign erythrocytes ' led to
the formation of antibodies which were mainly:of - the macroglo-
bulin type at the beginning of immunization, while:affPr 14opeated.
imMunization a large proportion ofantibOdiPs was formed. with
an electrophoretic mobility of gaiiima2 ana a sedimentation -
,
constant of 7 S./15, 14, 8/, this -time corurse tn. the rormation,
of antibodies of the gammaim and gamma2 0.pe was demonstrated
STAT
In further types ?;Of corpuscular and soluble protein antigens.
/2/, ?
. 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 formea.
togetherfin 'the lower vertebrates /16,1 7/ and bytthe fact ,tha't
,
. a similar time course in the formation of the twe types of:anti--
. bodies wad found in the period of immunological tmaturity 11/. .
' Little is yet known of the serological propertifes Of different
types of antibodies, It has been Shown that macroglobulin anti-
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16 o,.i /.J.s,
...? than 7 ant pies . of
4.
.... r . . ':.'g ' ?
,; '
4. essentially tricrfeetiVe in the'haemaggination reaCtiOn.
, ? ?,. ? . , ' -
.in whicli.7Q0',.times less Malroglobuilthan,7 S'antibodies are
,1
,1 .., .,. ' '.' r? , ' .
..
'necessari,,te'-produce the same eff6'C,t:/9/. These results,
:however', tOu6b.'-on1vnli:*=.imall partef antibody activity and
, ?
it would be most importdra to carry out a comparison and
different serological manifestations on reaction with antigen.
.We have attempted to make a comparison of the activity and
specificity of antibodies-Of the macro8lobulin,and 7 S type.
Chickens were used as the source of antibodies since they are
very good producers of antibodien to soluble protein antigens
and produce macroglobulin antibodies in relatively high amounts
/],, 4/0 In addition antibody formation in chicken, ig very rapid,
a high level appearing even after primary immunization,
kgroup of chickens /Leghorn.2,weight 1.5 kg/ were ammunized
with one dose of 50 mg, of humQm 'serum albumin p-azobenzoic acid
,
/p.;ABA-HSA:/9/,
r..
tibodies to protein'carrier determined bY? the haemagg1U-,
? , !
tination.Of HSA sensitized erythrocytes appeared in all chickens
as soon. as on the third.day?after immunization,, whereas.ani-!
bodies to hapten,.also determined by haemagglutination, did not
appear until the seventh day after immunization and.then.only.
:
in low titres similarly as described by Geld and Benedict /7/..
,
The birds were exaan8ulnated on the seventh-day and the types
of antibodies present In th6.se.rum determined by ultracentri,
1
fugation in a sucrose gradieht and by haemagglutination of.the
separate fraCtions, Ii accord with the data in the. literatue
/1, 4/ antibodies to HSA wore of both the inacrogThbulin and
7 S type00n,tho ,OthQr:hp,nd, 4ntibodios to hapton wore exclusi-
voly of the. Nacroglobulin .typp./Fig.1/..
? ??
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immunization, antihapten antibodies were of the 19 S type, in
a further experiment we attempted to' determine whether anti-
bodies of two physico-chemical types can be formed to one
determinant groAD, 1,e0 haptenic group 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 chickens was immunized repeatedly with
40 mg. of p-ABA-HSA Given intravenously at weekly intervals.
Blood was collected on the seventh day after immunization before
giving the next immunization dose. As evident from Fig.2, anti-
bodies to haptcn in the first two collections were of the macro-
globulin type and after the third,immunization dose antihapten
antibodies of the 7 S type appeared, so that after further immu-
;
rdzation antibodies of both types were present in the serum.
These antibodies to hapten were demonstrated by haemagglutination
of p-ABA, bound by azo-linkage to erythrocytes;,In this system,
in addition to the actual hapten, the amino .'acids, with which
diazotizedp-ABA reacted, could form part of the determinant
group, whereas the other carriers, ie. HSA en immunization and
erythrocytes on detection, are quite differente-This denotes
that both types of antibodies, macroglobulin and .7 S, are formed
against the same determinant group and thatjlapten alone or
. possibly a hapten-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 in,rabbits, similarly.
j
That the two types of antibodies have the' same specificity
does not, of course, denote that both must have the same immuno-
chemical properties, i.e. an equally large combining site, the same
space configuration of the combining site and therefore an equally
,
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4 0?
firm binding with antigens Unfortunatoly,'we could not used
antihapten antibodies to obtain more details about macroglobulin '
combining site because these antibodies occured 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 seliological 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 salines The sodium sulphate was removed by
filtration.on Sephadex G 25.
We worked with pooled sera from.three-4roups, of chickens,
ises taken after primary. immunization, after secondary immunizat-
ion and .finally with hyperimmune serum The'birds.were immunized
intravenously with 40 mg BSA at?monthly intervals, .hyperimmune
serum was collected after the fifth immunization,
e
In accord with the data in the literature, in all three groups
we found haemagglutinating antibodies in bpth the macroglobulin
ahd 7 &fractions, Figs3 shows the eluates 'Of, the primary and
hyperimmune sera from the Sephadex column and haemagglutinating
titres of the separate fractiOnss It is evident that in the primary
sea most haamagglutin ting activity was present in the fraction
eontaining 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 presenWof cross-reacting
?
antibodies. If a comparison is made of the titres of haemagglu-
S TAT
. .
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tination of erythrocytes sensitized to BSA and cross-reacting
? with'HSA in the separate fractions of the eluate, it can be seen
that antibodies cross-reacting with HSA are Mainly presont in
the fractions containing the macroglobulin antibodies whereas
in the 7,S fraction their titre is incomparablY lower as
compared with that of anti-BSA antibodios,
Antibodies were further determined by quantitative preci-
pitation. In concentrated fractions of antibodies of 7 S or
macroglobulin type the precipitin reaction was made irL0,15 M NaC1
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 sc5 great
that' they did not permit the determination of amounts of antibody
of less than 20-30 /Liz Ab/ml,
The Precipitation of antibodies of the -7 8 tyPe?:was posi-
tive in: 1.5 M Na.C1 in all three pools of sera inveStigated. In
0,15 M NaC1 the reaction was positive after the secondary immuni-
Zation and in hyperimmune serum.? but not in the primary :serUm..
The amount of antibodies precipitating ' in ',0.15M NaCl or in. 1.5 M
NaC1 varied so :that the relatively highest:, amount of antibodies
precipitating BSA at 0,15 M NaC1 in the mixtureof hyperimmune
sera similarly as was described- by Benedict 'et al.' /4/. .This
also explains: the increase in .haemagglutinating Antibodies in
fractions corresponding to Atntibodies of the:? S type during
immunization, since only antibodies precipitating in0,15 M NaC1
?
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14/ are effective in haemkgglutination.
A' compaiiSon! of the amount of type. 75 antibody precipi..:,
1
:
tatig in 0,15 M Nadi with -haemagglutination titre of that
fraction showed that the sensitivity of the haemagglUtinatiOn
reaction is within?the.usual range of sensitivity for passive.
haemagglutination, 'If we compare the high haemagglutinatin
titres of antibodies of the macroglobulin type With the negative
? results'of quantitative precipitation /What means that the amount
?of these antibodies was less than 30 /ug Ab/m1/1.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 amtierythrocyte '1' antibodies, Alen)
1M
?
Groenbury et al. /8/ found that for producing the same haemato-
lytic effect, an incomparably smaller amount of macroglobulin
antibodies is needaitham in haemagglutination with 7 S antibodies.
We next attemted to determine the specificity and binding
power of haAnagglutinating antibodies of the .two types. For. this
purpose we used cross reactions with HSA and inhibition of haemag-
glutinatiqne. Inhibition was .studied. by adding 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 thred tObt'.
sera, It is evident that in crOss reactions the ,intibodies of the
two types differ considerably? Antibodies of the anti-BSA macro-
globulin tyPe' give cross reactions with HSA.sensItized
cites to a high degree in all three cases,: In: the Case. of anti:.
bodies Of. the 7 S - type cross rodctidris were essentially less,
these antibodies showed a far higher epecificity to 'BSA.
-Similar differences were displayed' in tho. haeniagglutination ift7,
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hibition regction. Macro131obulin antibodies were only inhibited
:by-homologous antigen to a small extent, .whereaS the reaction
of the '7 S type antibodies was almost completely inhibited by
,
homologous antigen. On coiparing the reaults Of ,cross reaotions
of macroglobulin antibodies ?from the samples tested no easential
differences could be found; 7 Stype haemagglutinating -antibodies
? on the other hand cross react in hyperimmune and secondary sera
less 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 iirtmunization, which could lead7to 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 less complementary ?to the ?deter-
minant group of antigen which would permit a higher degree of
cross reactivity. The lower degree of complementarity, could
being
be due to the combining site smaller ,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, leeedifferent8 in, the combining site of antibody
were true, this property would necessarily manifest itself i?
greater dissociation of' the antigen-antibody complex, This
corresponds to the result S of the inhibition .reactions which
show that the system macroglobulin antibody;-BSA-erythrocyte
is only very little inhibited in the presOn.ce of free a.ntigen
which is evidently due to the high dissociation of the antigen-
antibody complex.
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Summarizing .Ou'r?r-e-sUlts- we can say that macroglobulin
z
antibodies have diffxrent properties 'from 7 S antibodies. Both ty-
pes of antibodies can arise to the same determinant group but
the, specificitvand firmness of the binding of macroglobulin
antibodies IS lower than that of 7 S antibodies.
References
1. - Banowiti,
- 399 /1963/.
qi:nger, S,J? and Wolfe H.R.: Jammunol. 90:
Bauer? 11)C. ,and Stavitsky, A.B.: Proc.Nat.Acadw'sci, 701,667
, /1961/,
Bauer, ?D.C.:: J,Immunol, 911,323 /19634,
Ben,edict,A,.A.; Brown, Raj" and Hersch, R.T.4,3.ImOunolf
90: 399 ,/1963/.
Hersh
91: , 795 /1963/.
6, Vaheyi.J.L.: Adv.immunol. 2 : 41 /1962/.
'Gold, E.F.,and 'Benedict, A.A.: J.Iffimunol.,89: 234H/1962/4
Goodman, H.S.: J.Inf.Dis 105: 69 /1959/,
R,T,0 and Larson, Ch0I, J.Immiunol*?
.
Greenbury, C,L., Moore, D.H. and Nunn,- L?.:A Immunology
61 421 /1963/4
Nisaonoff, A. and Pressman, D.: J immunol. 801 417 /1958/'. :
11. Rihal Symposium "Mechanism of immunological tolerance
-Prague 1961..
12. Riha,
and Svi6u1is,!A Folid ticreb1o1.9: 45 /1964/.
13, Smith0,R.TO:In Ciba Foundation Sympw,"Ce11ular Aspects (of
'Immunite, Churchil, London 1960, p3.4$41
14. Stelos, P. and Talmage, D.W.: J.Inf.Dis. 100: 126 :/1957/.'
15. Talmage, DW., Freter? GO., and Taliaferro, W.00; J.Inf.Dis.
981300 /1956/.
160 Trnka?. Z. and Frank, PItt Folia miorobiol, 5* 374 /1960/0
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?sulphonated Anti-dinitr' ()phenyl Antibodies.
Some.Specific Features of the Interaction between Isolated-
'," ,
?.
yr H and L Subunits.s.
4-
73?ranZik, 0.got?frnek,. L?
./.
1)opart4ent of Iirh)unology, Institute of Microbiology, Czedhosloval:
loadcmy of Sciences, Prague, Czechoslovakia I.
..cEhe exl.etende of an active site on the protein Molecule
? composed of several types of subunits gives vise to the queE.,tion
as to Whith t9?pe "is the ce.14rier of the actile Site' "and.who:ther
it 1s one type or whether more types particiPate, If .wq con-
_
sider, antibodies, which are known to made up of V:70' types of
subunits, H and L, it is clear that there 'are three basic
. ?
? possible? answers, to this question. The carrier ,of the:aotiVe
92.n be subrinit H, subunit L or both of these? subunits can ?
Pticipate in its formation.' /11Lese basic alternatives comprise
within themselves further cases extiressing the possibility of '
: ?
the interaction of several subunits of the same typo .-or the
differentiation of the role of- carrier ? of specificity and acti-
vator itisnot difficult to consider these posSibilities?.
and to disaus. , _their consequences', but; su:Jh. considerations
do ndt of themselves lead to ,any advance in the solvina of the
problems- no i3ossibility can a priory be designate-d as impossible
?
In 19 61, on the basis of the electrophoretic. analysis of diffornnl-
guinea pig antibodies, Edelman et al /l/.ecpressod. the .opinion
that antibody specificitls is given by the chain, This opinion:
'still.' not supported experimentally by testing activity, was
maintained by Edelman and Benacerraf in a .further publication
/2/ in 1962, in`which other possibillti...?,atrw discussed; #3.64
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Fran6k et al.-2
the interaction of two different or siniiar L chains, Before -
t11:1.i in 1902 Porter /3/ has already published ,experimental
renults plricing the antibody active site,into the H Chain, Cur
ioic published-at the beginning of 1963 /4 7/7- however,
1-7:?1_nted to a different conclusion. No appreciable activity'
:7.'ound in any of the isolated. chains but .reappeared 'after.
r.-.1.:-.1n2; them.. On Mixing H and L Chain's, the activity of anti-
bed.'_es of different specificities was also covered 'but: only
rtically and the hybrid possessed. a specificity correspon:V.-1,-,
to the H chain used. From these results ze reached the conelu--
f..cn 'chat the Paticipation of both H and L.chains is needed for
the 'formation of the active site, and that specificity' is actor-
by the H chain, whereas the L chains may .be partly
in their function 'by, the L chain from another antibody. A verr?
1.-portant finding was that isolated H. and 1,- chains are capallle
?
of 7:iutua1 interaction even without the formation of covalent
%ords, The mddel of equine antitoxin used, holever, was not ?
ideal froin all aspects., Although therecovered activity. was
eg.l to the activity of the preparation which was subjected to
no same:treatment without separation of the chains, this astf..-
vity very low, maximally '5% of the protein in the sample
*.:as active. Apart from that the determination of activity by
r?"--la of an tlimmunosorbent did not permit the extension of t1-1_
to the measuring of the physico-chernical parameters cC
For the further study of this problem we chose antibodicr
to the dinitrophenyl group, wh,ose preparation holds 'out the
possibility of a high Yield with a relatively narrow specificity
Zig animals-bulls and pigs - were chosen as immunization obLeci;s,
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Frank' et aJ - 3
a. 2.arge series of experiMents and partly because perni.t-
? r!cing with antibodies from one individual So that soim aspc?3..:
heterogenicity can be excluded., The imuniing antigen 1:1 a 3
ma- 31obulin , modified to a high .degree by 2?4-dinitrober-?eno
-ulphonic acid /8/, was given in adjuvant. Bulls were 1:-;-.7nun:. ze-1
-7::.;h pig gaFma-globulin, pigs with bovine samma-globulin. 7n
--):_zs a single immunization with 100 mg, antigen 2 - 3cweelt..s
''ora sla.ughter proved satisfactot7? The concentration of qn t
'7:D?'.5.es. obtained was -0,1 - 1,0 mg,./r1.1- In bulls. an antibor:I7
trtn of - 1.0 mg./r21, was attained-two weeks after c.a.')
of 200 rng,antigen animal. Repeated doses of antigen-4_1iT .
,-77 ? -
P ay s DrodUced approximately the same increase in the concy:)-
.%rion of antibodies so that blood could be collected;;r.s.T.
contsining antibodies in concentrations
suitable, for isolation,
considerab.ly modified .the method used by Farah et'al,
/9/ for .the ? isolation of antibodies ?After absorbing the an':
4:17.e carrier protein, antibodies to the. hapten Were prec!...-;L-
ted by diriitrophonylated garrne globulin and the washed pro.-
' tate v:as d.issolved with 0,05 Lidinitrophenol in 0..1 T ph0F.?1.--.:C,
buffer, pH 7, containing 0.,5 Na.C3? The solution containing
? bigen and Jaapten was applied. on a D.7AE-Sephadex colum-L
with the ? same buffer without dinitrophenol. Antigen wr_s
firmly on the top of the column whereas antibody
..71,.thout adsorption and was thus separa.ted from the dinitrom:...1
:Lose zone moved down the column very 81ovily0 The..antibody
on the DEA,E-Sephadex reached up to 755 of antibodies determl.ned
in the serum, by the qu ntitative immtineserbent method,' In other
cases the precipitate was. dissolVed with the buffer which
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/AT
?
?
' ?
Frank et :al., -.4
1 rc 1074 M .-DIIP-lysine. Antibody was soParated from hanten.
:71,1 antigen in the same vay as fram'dinitrophenol No7Iovelr.
t vas only possible to remove excess hapten, a portion o2
the '7:-D111"-1ys1ne remained bound to the antibody,
The chemical character of bovine antibodies is.? S gamma-
:leb.ulin as determined by starch gel eler.ltrophoresis and bY
'-71unoe1ectrophoresis, At the same time the *extent of oloctro-
heterogdneity is less than that of 7.8 gmmia-3bt.
T.'; displays to Indication that the antibody mild be pret.,on';
170 types, as vas. reportedv, for example,' in guinea pics /10/.
The average association constant of tho interaction of bovfrc
with E vae vithin the range of 105
:113 vaAous pr'earations,.It vas determf_nod
r_librIxlm dialysis and by polarography The polarogmrel-IM
of. dots7mining the association constattts er antibody to
diz.n?ophenyl group was sound very useftil tH.DN11-1r-'.ne
7. 7:311 mommreable !?olarographic dotble-vave, By adding
anrlyeed mi::ture the height of the-E,,DNP-lysine
do elrectly proportional to the concentratiot,of free
17PI.no &DNI),.lysine being not rogistered,polarograD;11cr/.-
27. TIT& great advantage of the, polarographic method is that
,-Ives results in the course of a few minutes..
The breaking dovn of the distphide bond vas .do:,o bra..
?
4 4-
1.11npnition, On the basis pf previous work on the reacAvity
cf the disulphide bonds' /11/ and on the nature of tlie
tnealatos. of limited cleavage of disulphide bonds /12/ t6
.4t a p11 86, i,o o. under conditions in which 8 disulphide bords
in the molecule are cleaved in pig gair,ma glob s tho secr
t.2.:arnative we selected S.sul-ohonatinn inT;
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- .t.4.u1.411.?Luz. JAJuul-Il1ucka.uu,2,0
,
and L? chains /12/, remain in the reaction-roduct, :Theeleet4O-
.phdretic .pattern. on starch gel, Shows that ih pig. antibodies ' there
? t ?
is no difference in 'the position of', !the tones rth the re1:a0.1.re
- ?
? .amoUnts'between nonspeci'fie tarrma-globtain 'and?-an. ?.
?
?? ? --;? :r? ? ? ,4,
. bovine _antibodies the tobility of ,the two types
' . ?,? - ?
?
somewhat loss than in .nonspecific,.
?
!
the: diffusion zone of.. the L chains is sMa..1..a.e.r addition, -
? .t ? '? 4 . At. ? 1.6 ?
t ? to. this there: is more incompletely split materiel. at-th0.q.e4Igt;r-
ing preparations /Fig.1/., S-sulphonation was ,a1?0 ? oarried out
?
"
--
inth
. 6 preSence, of excess of dinitrophenol? brDNP..Lysili, As
i
?
seen from Fig.2 the hapten bound on thp,..antl:bodyl#,,es.'not,6?ffect '
the -Splitting of -interchain'distilphidebondsc? '.11).V.62.eCtrO0-4setic
?
pattern of subunits is thci. same as, after ','S,!"'SuIphanktion,,- in the
,
? , .?
abS ?nee of hap-ten. Fig,3 confirms that :rieither ;#1' :4-si.110-ionktiOn
at pH 5,7 is the character of antibody 1,6buniti-- ? ,
,
.:aiffcrent..from the, character of su'bunits .of ?nOnspk.6c4iric. g
_
!' ?
. ? j
None of the efectrephoretic-patteribtadtn.:Iiireti".,Starch,
' ? -
4 S.
- , ? ?
? ?
gel at 'pH 3 - 4 showed banding of rthe L cha?,in
_Which were described by Edelman- et 'al,. /1/.'.1.,-1iipuret'illinea:7,13ig'
antibodies. Banding did not appear- in ?bovine- oil'ag antibodies .
4
'even when they had been subjeced to reductio ji 'and ,4ikylatfoii.,-
r "
exactly accOrdsing to the -procedure ,described 1y Edelman and
Poulilc /13/. The large amount; of. antibody, from siiiJ bulls made,
? 4
i-6 1.)ossib.e to 1-exatnine theheterogelAity,' ot, ..cha,ins- coming trot
diqeren-t,i'ndividual; by electrophoreSis "in stirohgel.6.t 15.4
? ?,.../? ,
/1.4/,...yvhere _sha'rp -baric,ls 'thatCri Chains
of -antibodies yield .a, sma11e? nuMberj of bandS' and tha:t Vo:or .0
,
hardly any. dif.ference' among .1.1141.,Vidllat
*
.? .?
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*Fran6k.et al, - 6
ris in the previous work Sephadex G-100 in 0,05 N fc=1,;
71:;11 6:1 urea was used for the separation of' subunits, Fic ?r:5
oho-:!'a the distribution of protein.after the Separation, of bo75.re
3-sulpho gamma7globulin and antibody on a Sephadex colurn,
Tho peak of higher subunits appears first overlapping pqrtly
7r1th the peak of the H chains, which is follOwed by a well
resolved peak of L chains, Under the conditions of separption
PT7 bound hapten was completely released from the prote17.
ed as a peak very distant from the L chsAns, The frao471c-iJ
containing subunits which wore taken into the experiment wel-
n'eo7.,od and concentrated with dry.Sephae_ex.G.05 The subun!_t3
"ere then transferred into (5.1 M bornte buffer pH 8,O en
a Sephadex 0-25 column,'1111 subunits'1:7ere soluble in this
buffor.
Binding activity was expressed by the quantity r denonr;
';he number of moles of hapten bound by a mole of prOt.e.fn;
cc-psrison, in cases not statea other7,v3e7.t7le molecula::
of p-..otein was taken to be?160,000; i,e; even In the case c'
:noirrted H or'L chains which actually havo a'lower moleculr12
r was always measured at a corcentration or free
lys!_ne of 1 x 10-5 M and at a tetperature of ':40C, The bln1:1-g
. .
o7:_olty of.nonspecific.bovine garza rlobulia and its?sub..='
'7r2,..esponded under the given conditions to. r = ThD
2n of nonspecific binding-capacity on the basis of weicl?.;
found to be' most suitable since the tinding'ear,acity of tc
o1:alns or from that of their mixture, bhe nonspecific bindj_nv
of 'c -Dn-lysine on bovine gamta globulin antibody and its
lylbunits was therefore eliminated from the results of mearurcri.:7,
_
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.1 ../, ' - : ,,, , '' , ? 4 '" .. *., ; / -1---;t
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.....
?
...
. : . .
. .7 : . ?? ?.'i??1.11 , -, ;,; , ',.' ..: ? '
' .,? ? . ..? , ?,' i
. .
- - k
'? ;1; '''; ''' - ? ? ?,,t :
,
Frank 'et'al.:-;'
. . , - ? ? . ,'.. -
units expressed. as r.
We first ? determined .the .value:?of'r"ktOr..nat-i\-ie eantibodY Ari.d
for Sf.sulphonated antibody without Separation of'`Ith'e, chaina.
. . ? v
..Table 1 shows that S-Sulphonated antibody-whieh,w.as 'subjec-ted
,
to the action .of the separation .thediu...it, tY,',eXPres-
.
'
,
sod- .as r; decreased to about ;one half of its -s
t , .? ?
'activity.. In further cwork ve invest?its.E',,e'a,hether there was ?any
?.
residual binding-activity" in the individual SU.ibUnits' 'arislbg
?. from ' S-sulphonation at p1:1 S.6 and 4.6 '... =.1ligher .'so.b..iill'ts "iwhich do.
4 -
" 1 ' ' r ' - .t ' , ". , ' ' , ... ..', 1 'y ".
not 'differentiate' One from .another1 onf.the'. Sephlda,'cOlpi..un were
*. ? a
taken together for; analysis, Ir Fig:6 the;-r - of', th9 . indiVidual '
-.subunits is 'demoted over- the .corrbspondlng-,ftabtior?m ?,..-...l'hq-.
?
?,:l ? ' . ' '
represent the ay. erac,re- of -at. qeo..s"-b." thre,e e#,,erithentsn'd,,4 ar'6'
. .
cox% re.dted for nonspecific, adsrptioni, Arriong tfie subunits. ot-,
Ssulphona,.ted antibodies, it is -only the. higher, '..subundshose--?
,
,, -k 4 t \ ?' ': 1 '
inding-9?ctivity' is preserved,aft,or separation in-cafibrd'eitiallle.
,. -,,, , ? ,
, . A
? ? rn ,.
degree. ',Free II ,and L chains' takenwith.outV.iriut4al... 9?.entainixiation
. -,.
, .
,. have no, significant binding activity denions;ti;a6,19'`-`,:uyi erit4,15:- ,
.
, . ..., i ',. r. ? '" ,. '
brim dialysis
, .
' 4
In several pilot tests we cen-firmod that a"ctivity was
covere'd; by. mixing H and L ,chaInS,. in,. the I .111e; in' .equiiit
antitoxin ,where it was first .bbSOV:e.d byifs. 1We thereforp;iinder.7,
took, a ,..study on the conditions and, quantitative ,relilti:Onsh
the recovery of antibody activity We sought: the;?top'clitaions;
... - ,
promoting the recombination of the subunits. - We h.ftie ,found. that
. .. ._ ?. .;
? t i , .. , ,
'
the ' final activity was not ,inf;uonced;by ,whether H -and L chains
- , -, ' ' J- ' - ' ' - " ,, ,` ' . 2"'-'" '? :-, - - -,.: -,t' ?
wereynixed in aid separating' solution;: after "neutra/ization or
even., afcr transferring the separate chains into bora'te :1Duffer,0r
. .
? Recombination cif the' chain's thUSpccUr a-'ne!it'rgi
? - ,
? e
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ne a os enc eA or u.re a . and the history of the' subunits before . this
tine has ,no significant effect Ori krbcombl.ntit4oni.-,
? ? , ?
The .average . of ip experiments. .showed th,athe:- r?eicOvored
activity corresponded after dedl*bl.ng'xi9PAPPO?fio- adsorptior
?
to r = O,68. If -we wish tO know ther7ield of,ireterithination
of ,chains we must -compare the recovered activity. with the-acti-
vity of 8-sulpho .antibodies- which were ,not , b1t;,1Z)C:bed. to. separa-
? ?
?
" r'
? ? - ?'` ?
Table 1
' ' r ? ' '
siaowp that pi,eparation? correspbnclin.g?to this
requirement pob.sesses r= Q.864?:Tho,:recoVery-of Ieirldiri 'Act71,17,1ty
, ? .
. 'after -separation and. recorilainatio. Of the ,Cha,;4# was thus:almost'
? : ; .? ? ".? ,:,?
80%. I, however, we wish to compare the ? recombined' ?
. ,
? , ?
complex ?with the, activity of native' antiladAyi., the recoVer..Y.61" ?
, ,activitY. ai;1OUt -40%.
'
' I A
We 'fnlice d ? H a.nci L yhains:in , differerit weight,,?keeping
L
the total :Weight ?concentrationrconstaritt If ,,the; chainSTrecombined
in '-stoichidmetric relationship' and united, wf,-th a;_beri,a, whOSe ?
. ? ,
. -
firmness was comparable' With the covalent bon& with which : they
Werc bound in 'the native; molecule,:the sactivity.'?f6und eirr
ekperimentai set-up would follow the broken 4auveshown ii
? ,
? 'the upper part of Fig.7 with a' Sharp ,maXirtiuni 'co?respondingto
, ?
e ?
the composition by ,*eight. of.;:lf arid L 'chains. IrCactual.tact. the
? ? . . , .
activity of the, mixture was .different as shoWri'411.-thts.:-.o.wer.?'part.
of the ng,7, The activity.'',hp.s no sharp niaximum bu 'is almost
-constant in a wide range oflratios of HI It. We shall- attempt
?
to give an explanation of.'. this lo-er,abolie:,,she.Of the curve : on',
?
-
Piga'? when 'explaining other findings.
?
a
. In our next experiments we iaxited,rto "eiUcidate the reie
of ,hapten in tho formation of comPlexot? ? of H and *L
? -
,the determine whether the active complex Of chaint:AS formed ?i41.).
, ?
?
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??
ro.ridli 'et ??,a1.0,
?
in borate buffer in absent? of hapten or only after ? its ,addit-
, ? ?,
?ion. It is. obviods ?that equilibrium dialySis.',. CLnriot.provide'
-
-?
bAl answer to this qt:lostion,,4: TheMeasureteilt_ofindrease in the
- -
binding -Of hapten by dialysis stopped after given. time ?inter,-
vals gave results which were. not reproducible7althoUgh we tried
?
? ,
to standadize all ,procedures,-Polar.ography iirOVed:'Much more
suitable hero, because instantaneous states' of, decrear;e of
. free hapten can be followed. We have investigated, the course:
of the decrease of. free hapten Which was - ac/ded:,,in a concentrat,-i
ion: of 1.4 x lo-5)yi on the one hand ? to the ' native,: antibody, on .the
other' hand to the mixture o f H ,and L chains, in 'borate .buffer pre-,
pared fresh and to a mixture-.which.had been.'left;-'to stand ,for
20 hours; We confirmed that native 'antibody binds -hapten. so quc-
ly that- at the Moment of ,recodding the firsV,datal, teseverru
?
minutes after .the addition of,hapteni:equilibriuM has already
b'een reached' and the height' of the wave of ,free ,hapten shows
:no :further change; The curve on shown'. ths.t. -e-DNP..-lys in
passes into bound form very slowly with recoAbinecr,cOmplezes
of It and chains. In our cOnditions it?took at lea.st 50 hours.
11,02pten reacts with a grater' iri.tial elocity 1th preparat-,
,?,
,,..fons' in which the chains were mixed and inCubated for., 'long -.?
period before the addition of hapten, We a'SSurae..-that..,this. finding
.P ?
SUElteStS. that Hand L complex. with a si5ecifio:s:etivo zitQ is
? ?
?
forted in the mi.xture of chains .before the addition or haptene
The determination of specificity 'can also 'be: studied on
antihs.pten:, antibodies. We cOmbinod chaind frem bovine 'antibody
. ,
,on-ti*.one hand.with:chains .of nonsgoOificbovine gmmnalobu1tn,
On the other , hand with .chains of -antibody to,, .the " ate: deter-.
-
minant group. No 'hybrid ? of Tbovine, and pig ^pa'rtibcidieS,
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.:koranek ?et. ai.
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, the.. mixture of bovine H. and pig LI nor he mixture of pig H
and bovine ? L, bound c.-DNP-lysine specifically; ',1:\To aspect other
tha`n this binding was investigated so ,that ,cannot ' saY whether
the? chains', reCgmbined at all or whether possible reconbine
was inactive. The activity? of, hybrids of Apcio141.c. a:nd
?
?
nonspecific Chains, are given in Table 2..: Hybrids' of tspecific
H clains'Ivith; nonspecifiC..L chains, also 7attain a, Certain s.c.t71.:-;
-, ,. .
, , ? . ..
-
Vity; even though lower than the complex with specific 1 :Chaii:.11,
1
'whereas in. the hybrid; of, specific L chain with nOnspedific H
4e
activity 'is ,not, significantly increased,
In, 'order to appreciate the role of the, in?iduai ch.411:s in?
determining .specificity and 5,.n the, formation of .t1:16 active
we
elaborated an, auxiliary concept, in which we condidered. alter-
natelv one chain as
a.'sore.of "inactive .rOrii- of the active ite ,
,
r . .
carrier? -:the -secf5nd 'chain ? as'. itS.,"activator",:This,7:cpncept
.?., ?
, . ??
emPoW'ers? us ;tb evaluate the activity loy; the .`quantiti'dis nr:H11?.nd?vri,
.
_.
'which 'represent the number. Of moles of t ? -DNP4?..ysine - bolind :to
..
a "Mol4T.'bt chai.n Considered as the 'inactive'. form,' It Is 107,000
?
?for 11 arid.,5S., 600 g... for L chains - in .this.,..ratio" the mole ,
?
-,:p.ntibOArt lee il6b, 000 g, 4s Composed. In 'ca..seS. where II] and L.
chains:are mixed, ,in. t49 ?same ; proportions, as preient, in the native,
?
, " ?
-:molecule, ..the quantities rH and rL are equal- to. the ? quantity r ?
calculated in relation to total, protein.
.4219 Fquantities rH Sm.& calculated ifor the data -inl'ig.rt
, . ,
;.inctease in direct' propoi,tion.. to the presence ofexcess of bhe ? ,
;II oh6nt. Cansid.ered as the "activator" in the mixtu' 'Ail:ado--
?
quate explanation fOr this finding is evidently the. 1..ad..,that:
.7- .
of di and L Chaihs cannot be compared n its
fiximneaS with ne.tiVo, antibody. since it shows considerable.,dip-.
?
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Dociatlxin, tt:ioildWa from thio, of coiaroo, ttathe:,ciaantity
t ? ,
Z' Is 'not qitablo for :expressing recovered activity Alince it wills
? ,
be dependent on the concentrations of trOtein' coppOnents and that
'it will., be, necessary : chooSe:.".a different, Way, of evaluation': in
further experinents. A confirmation Of, this :explahation can only .
, , - ?
be given ,when data on the -binding of hapten on complex H. and L
chb.ins 'txi a wide ,ra.,nge of protein concentrations, Will be
able, .The:data rom" duch ?.e.,4eritherit wbuld 'also, :.decid.e
hOW. mUoh",of the.', decrease of activity found in S.:sulpha, antibocli.es
:
or in ? complex of . antibody chains' ?in comparison "with",:native-Jnolecu-
. ,
le /see Table 1/ should'. be, aseribed to . irreversib1e": inaetiv'ation
c `-?
,and Chow much. is;' due to the fact that in the caSe of the ,Complex.
vie are dealing with -a multiComponent: dissociating ?Systeir10.: It will
e
,
s...so be necessary to :obtain experimental ?data on sthe basis of whicla -
a standpoint -could be : taken up towards _the,. alternative 0.#1anat-
4.
. ? ,
lop of theiparabolic .shape of 'the ciarve'dn.Fig*7..i that -various
?
4 4 .
active compiexeS o-g ,.and L chains are ford in ivhich-the*".iatio'
H ':,-L-iS pot' in all c? tii same as .111'; the. nattire
o' iiifferen,t ' role :of the two chains in; 'deteriiiining'
,
?
'cannot ? be: displayed- examining "the combiiiation 9f "chains
originating from the same antibody. This question can only be
- ?
?
decided.,-by..detofimihing whether and to *what degree sOrhe chain. :
?
can be:.. substituted by a chain from "nonspecific..
The diffei-'ent :role vas best displayed where 'excea's' nonsPeCifiC
'chains :Were ,addecV /see Table' 2/, These 0. ekperiments'Show that the
. .
, . ?
,
chainj cansidered to ,be ,theinactive form" can- be .a.ctiva.ted' by
Orcess. nonspecific L chains to a, greater ?degree than by . an
,
adequate'fcitiantity of ,,specific.:, chains,' The reverse. conception, 'how -
. .
ever, fails': I .chain as ;the "inactive form" : not "activated"
;:by no,nopOifio.. 4.1dhains?). Increased activity could 'clenl'Ondtra:?:
-
? L STAT'
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creatiorl. of a c..oncept of H stibtinitt as '`.11,iriactilie 'forme an'd
s :1?,"
chains as '"a.tivator", fOr the illustration of the .Uriecfu'al-iiritortan-?
? .
ce of H and T,.ei*ins in ;the active :Compikt. doe8.i4t. tie 9,11: that
?
consider!. the, Synthes,is' of ?: ant ibodY,1 take'.plaae
?
form.,Ths sighifia.nce of dt,chains ' can ba compared with the
.significance- di ,inactive forms -9.41. other ?lbfal idallvactive Sub-
, .
"stanbes'. The. significance of the chain is *best ,understood bytho
,
term,lia.C.tiVatorli,:thGe. effect Of its additior). can' ..best i-)0:: comparOd
?
Oilv,ith the 'actOation, Of, enzymes , by: the addition ..?_):f - various',.'c '- ?
, . ? .
'.enyrries; 'metal zi.?Ons etc 9,'It remains 4 to 'ejcplain w14-1.y the;:ef'fecth
,
it 4,4 ?
"
,nona.peCifiC gamrna7glebulin are not quantitativoly
identitcal'Vi6th the t ffect of chains of antibo,tr_q. Chaii:),..s, are
:undoubtedly hete1Og.3neous. It 'appears that the extent
;hOteregene-ity? of ,the L chains of antibody is less. than :that :.
;
? ' ',..... ? r ? .? ? ?
of the L ,ch.dinS'46i,.?rionsecifia '?garo-rrla.-Iglel.pu4.'n 4. Let. 'P'sa ???.9..8 a urief
..-: that; ,thee.' billty' to activate u,:specific .11. Chains ?-1:6.1a:...pre2pe'rty ?
, ..- ? - .. ? .
, . .. - - ; ?
:,..0., ? ..,:-.., ., .. -.' .. .,.- ,
:
of '.-onlr,-- cei4tain. L, chains 'from 6,, whole 'slat which is f,ound...11.n.':
nonSpe.ciTia :gaMma-globulin or that this ability is not iareset '
in all 1.4:. chains tin -"the..saMe 'degree 0 At the 'same, ,tii-na ?it- need,.
.;.,. -,7.--' '',.. ', ,:'''' '''-.. :! '.. .' ' . '' :': ',': .',:. ?'',...;: .,:-',.,..':: .-
..not 3oe'? diatiirie'd :that the Tability to ? vactiva'te f.` . the it...;:chaiii of ?
, . ..
tk? ?`91.,..b6...ip..?ap:ogificit4 is nec'eabarily 'related to si,E._2ifl:o1ii ,
. .
. 7 ? ,I3 ? t t
t . : ' 4 3 I ' 4 ? . . ? 1 ,s3
3 ?
? ? ? ,I.
to', hapteni :The' qabtiVatihe effect of ,L chains no
' of2iispeolfi4. .
, . ,.
gaiiitna-globulin :d.,,,. therefera` less than that of L:, Chain-S- 8.?-:,::
. I . . .?. . . i ? ' 1
1 .
antibody-. If added inexce s, however, ".activatioiin takes' plac'e-
, . ..
. . ..,, ?. , .k.
. ,
G the ,,s aine. dr' to a ,Shigher degree.
? ' , :. ,:,' I , , . . ( l . , 0 ,e
- tleillg%. the model of ;.ii:tiljody'.._to ? the din' itrOphenyl tvb,t,ip, .we ..
. ? . ? ., ,
.. ? - ?
.. ? ,.
...succeeded in .60-Ifirming all the findings whibh vve'? mere .tlie;first
,
1. ? establish', on the model of -equine antitoxins We confirmed - that
? isolated '-sub*its 11..and'L do- :not, display, any appreciable, binding
activity, arid', that residual, binding ..activity is ?only, attributable'
-. : ' ' - ? '
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_ - Ullid400U1CS. T.:10
that the bindinG activity is recovered by mixing H and L c;1%;
aI'c,hough restitutilon of the covalent bond between them did 1,-;L;
was convincingly confirmed. Finally, it vas confi.L.mc,f.
the H chain plays the decisive role in determining the
city of the recolZeLned complex.
The model voking with two easily soluble high molco
=7:Do:lents /II and L.chains/ and a low moIecUlar hapten
poseible to study intoractions by?physico-chemical methcdc
;.s found that the in:4_;aaction of H and a L chains takes per,
a neutral medium even without the presence of hapten EJ
slewl-y that the rec_ching of equilibrium is a question of ds4e,
Findings on the role of the chains in determinirig specifitita
tupplemonted by a number of findings,-L chains obtained L.
nonspecf.fic.garai,a Globulin can produce the same "activatie.L
o2 the antibody H chain as L chain from antibody f4? the non,??
fie L chain is added in e;:oess.? On the other handl. the L cia:
em antibody calnot "activate" nonspecific H Chains ellen
,..aded in excess,. Nor did we succeed in obtaining active hybr5is
om the chains of bull and pig antibodies even when the anG.
Lctnes wore di-noted against the same.doterminant .ccup. Th
ns,w'data led us to the hypothesis of the mechaniEM of intr-
action of H chains oarlving po'6ential specific binding actlY
dith het4roGeneous mixtures of L chains frOm wach Only sc,Lo
provide an active cemplext, It would appear at the same C.2.:o tl.at
coilplex'is considerably dissociated, Sufficient .variants
. ?
of the el:periment-s have not yet been made to confirm thiz-,
thesis but ths results obtainod to date show that S-sulphe su---
v_nits of antibody to the dinitrophenyl.group are very ,suitable
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, for further study and wellppe,that we "shall ,sueeeed in detoctin
a series of further properties of antibOdY4ubunits'oh this model.
References
1,. Edelman,. G.M., Benacerraf,, B., Ovary,.
Proc.Natl0Aead.SO4 47:1751./1961/0.. -
?Edelman, -G.M, and-Benacerrrg, Proc.Natl.Aead.Sei.
1035 /1962/.
. .
.Porter, RR: The structure -of gamMa-g1obu1in and anti-'.
-bodies..In Symposium on ,Basie Problmms of Neoplastie Di-
teases: Gelhorn 'and E, Hirshberg Eds. Colulibia Univer-
-
.sity Pr,ess.1962.
Frank,. F. and Nezlin;lki.S.,'presented by .T.4terz1; p. 74
.in Immunopathology, IIIrd International Symposium P.Grabar
,.and P,.A.Miescher Eds.'Bsel 19e3
?
:Fran6k, F. and Nezlin, R.S.1' Fo1i'1pierob1o1.. 8: 128 /1063/.
6. Yran6k,.F. and-Nezliiv Rip.: Biokhimiya 193,/19P3/.
Fran6k? F? Bezlin, Folia Mierobiol.
8: '197 71963/.
. ? e'
. -
"..Eisen, H.N.; Kern, M., Newton, W.T.,an,d.Helpreieh,
. J.ExpoMed. 110: 187 /1959/.
.Farah, F.$? Kern, M, ?Lx1).Med.:112:.
'
1195 /1060/... ,
'10,: Bonaceri-af, B.0.0vary?,..R.4 Bloch,:K.J., and Franklin:, E.C.
?
'
J.Exp.Mdd. 117: 951 /1965/0
?
?
11. 7.7rank, F. and Lanka, V.: ,Collection Czechoslov.Chem.Cem-...:qc.
2.82 24511963/i; , ?
12, ? Frank, F. and Zikan,?J.:. Collection Czechoslov,Chem.Comwu,
29: 1401/1964/.
4
,
'.. ,
, t
? : '1 ? . ?
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' STAT ?
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-
',Fran4k 'et-
36 Edelman GM, and. Poulik, MoDo 0- 0Exi3'Med, 861
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?
I ..?? ? : A, ?
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..L.JutLufluuto
Fig.l. Starch gel electrophoresis of bovine and pig antibody
and gamma-globulins-and their derivatives.,
Compo6ition of.buffer; 0.05 M:formic.acid 6 M.urea., bovine
gamma globulin,-2 S-,sulpho,bovine anti-DNP . antibody, 3 .
S-sulpho bovine gamma globulin, 4 - S-sulpho: pig gamma globulin,
S,sulphO pig anti-DNP antibodies, 6.pig gamma,globulin.
F4.g.2., Starch gol olectrophoresis of bovine WO:a-globulin and
anti-DNP antibodies S-sulphonated
Composition of buffer: 0.05 M formic acid, 6 M urea,
gamma-
globulin, 2.- antibody, .3 antibody-S sulphonated in excess
? .
?
dinitrophenol, 4 antibody S-sulphonated in excess L-DNP-
? ?
lysino.
'Starcil 8 1 electropheresi6 of bovine:gamma,,;lobuliri and:
anti-DNP antibodies S-sulphonated at pH 5.7.
CompositiOn4of. buffer: 0.05 .Mformic 4.cid,.614 urea.
A
globulin; 2 r.antibody, 3 , antibody S,sulphoi.lated in, excess.
? Aitnitrophengl 4,- antibody !,.Sulphonated 'in excbss-E-DNP-
gamma-
lysine.
Starch gel electrophoresis of L chains of gamma-globulin .
and antibody..Composition of buffer: 0.035 M glycine buff6r,
0..8.8 8 M'urea? 1 - chains of nonspecific lo6-siine gamma.
globulin
21 3, 4 -'0ains of isolated anti-DNP'aritibodyfrom
three individual bulls.
, ?
-Fig.50 :Chromatography of Z-sulpho bovine .gamma globulin and ;anti;
?
? ,
DNP:Antibody on Sephadex.G-l00.
Medium: M formic acid, 6 M urea. Abscissa: volume of-aluate
.?
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Frank et
Ordinate: optical density at 2537 ,a /registered by UvicOrd
- Crosshatched area denotes fractions pooled'.. bl.ft6rent sub-
units denoted by letters 0.
.Activity.of subunits of bovine anti-DNP antibody
S-sulphonated at different pH.
, Medium' for separating subunits OO5 M formiO "acid, 6 urea,0
Abscissa: volume Of eluate. Ordinate left: optiCaldenSity ,
at 2537 a. Ordinate right: value of Upper ,part
.Pre-
paration -0;sulphonated at pH 8-0*, lower part Preparation
'S-splphonated at pH. 537. Activity expreS:sed: by qua4ti s
of rare depicted as crosshatched coluMnS and giVen
numerical values appended to the separate ,Subunits
Fig074, -Activity of different mixtUres,Of H and L',subUnits
-
bovine anti-DNP antibody ? Abscissa; percentage' pf Subuilit
in mixture,. Ordinate:- activity- ej"?.-,preS sod aS
_
Upper, part, hypothetical curves correspohding .to ideul.-caSe
-
Of , VerY firmly bound H and L Chains in s toiChiometrie
:coMpleX, Lower part. experiMental curve'?
Figej3 Poiarographic registration. of reaction,
f
with complex of H and L chains' of bovine antii,DNP. antibodY
, Abscissa: time interval from miXing of 'protein with hapten,;,
011dinate : concentration of free E-DYP-]ysine Total 'amount
of protein in experiment 2,3 111F - haptend4ded immediately
after Mixing HI and L chains, -2
mixing H and L chain
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Franek ? al. 18
Table 1
Activity of bovine anti-DNP antibodies
d their derivatives
No. Method of
modification
Relative activity
Nat 1e
1.77--100
S-Sulphonated?
/pli 8.6, 20 hour./
a/' dissolved in
borate buffer
b/ dissolved in 0,05 M
formic acid with 6 M
1,40
79
)area and transferred
after 20 hrs, in borate
' buffer:
0.86
49
+After reaching equilibrium with 1 x 10-
STAT
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? Franek et al. - 19
Table 2
Activity of mixtures of bovine an DNP antibody and bovine
gamma-globulin chains
+++
Type Concentration :Total r++ rH+++
rL
of + in mixture ;concentration
,
chain mg/ml of protein-
menl
0.50
sp -
Lsp 0.50
H +. 0.33
sp
+ Lsp 0,17
Hsp + 0.33
0.17
Hsp+ 0.33
+Lg 0.85
+? 0.33
+Lsp 0.17
H + 1.00
+Lsp 0.17
0.50 ?
0.50
0,50
0.50
1.18
0.00 0.00
0.09
0,58 0.58
0.34 0.34
0,36 0.86
0.50 0.11
1.17
0.06
0.09
0.58
0.11
0.14
+ 1181;$ Lsp - chains of antibody H L g - chains of nonspecific
g
.gamma-globulin
++ corrected for non-specific adsorption
+++ rll and rL - represent moles of -DNP-lysine bound to
107.000 g of Hsp chain and to 53, 000 g of Lsp chain
respectively.
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For discussion in the topic IV.
Possible Relation between Some Results of the Study of Delayed
Type Hypersensitivity in Tissue Culture and the Mechanism:of
Antibody Formation
J.Johanovsk? J.vejcar, J.Pekarek
Institute of Sera and Vaccines, Prague, Czechoslovakia
Antibody formation and delayed type hypersensitivity are
usually considered as two principally different immunological
mechanisms, although potential relations between them are often
discussed. Most frequently the possibility Is considered that
delayed type hypersensitivity represents a certain stage in the
development of the mechanism of antibody formation or that the
state of delayed hypersensitivity is evoked by the binding of
specials, unconvential antibodies onto cells. Both phenomena ,
/mechanism of antibody format3)on'and delayed type hypersensitivity/
are mostly studied separately, without paying sufficient attention,
to the fact that both these mechanisms usually exist:in the.-
sensitized organism simultaneously and that they both can parti-
cipate in its immunological reactions.
In the stuay of delayed type hypersensitivity various methods
Were intensively developed in the past few years for the demon-
stration of this type hypersensitivity in vitro; mostly by means
of migration inhibition by specific antigen Of cells frot a
sensitized animal. The importance of this reaction its specificity
and reproducibility are beyond any doubt noWadays. ?
Nevertheless, one basic fact should be kept,in mind. In
the experiments af this type the reaction of the ?cellular poRulat-
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Johanovskl. et al. - 2
is obvious that only a small percentage of the cells present can
be really sensitive to the given antigen. It is after all, un-
thinkable that in an organism hypersensitive-to several different
antigens each of its mesenchymal cells could be specifically .
hypersensitive to each of these antigens. This conclusion logical-
ly ensues from all we know at present on the basis and the manner
of the development of immunological reactions.
Recently direct evidence has been brought forward for the
correctness of this view. David ot al. /1/ have demonstrated
that mere 2,5 per cent from a.sensitized animal mixed with
97,5 per cent cells from a normal animal are capable of elicit-
ing a state in which the whole.cellular'population reacts on
the addition of specific antigen as though it. were taken from
a sensitized animal. It is evident that the migration of normal,
nonsensitized cells was influenced as a result of the reaction
?
between a small percentage of hypersensitive cells a.nd the ani-
gen; the most probable explanation is that in the course of the
specific reaction of hypersensitive cells with the antigen sub-
stances posseping pharmacological activity are released which
influence the behaviour of the remaining normal cells. We too
arrived ut similar conclusions on using another experimental
approach, i.e0 when cultivating simultaneouslj? two spleen frag-
ments, one from a sensitized and one from a normal guinea pig, in
a single cultivation chamber in liquid medium /13/ Our results
indicated that on adding specific antigen identical migration
changes occur in a part of the experiments in thefl normal' gragment
taken from a hypersensitive donor. The only possible explanation
of this phenomenon is again the presumption that some pharma-
cologically active substances penetrate through the liquid medium
. from the hvnArRi,,,rqi-Hup "Finc, nnrmn1 frnrrninf!
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et al. /9/.
The mentioned results are in conformity with the general
hypothesis presuming that the reaction. of hypersensitive, cells
with antigen induces the formation of pharmacologically active
substances /mediator/ which elicit, through an irritation of
cells, :the actual manifestations of delayed type hypersensitivity
15/.
The second important point is the question, whether the re-
action of hypersensitive cells with antigen in vitro. always leads
ohly to the inhibition of-their function /as it is generally
fbelieved since the experiments by Rich and or whether
it can manifest itself sometimes, on the contrary, as a Stimulat-
ion., Evidence can be found here and there in the literature on .
the stimulatory action of antigen on cell suspension from hyper-
sensitive animals /2, 6, 14, 15/. Also recent-studies on the.
induction of mitotic division in the tissue culture of leuko-
cytes from tuberculin.sensitive subjects following the addition
of this antigen /7, 8/ may be a certain reflection of this fact.
Nevertheless?'most authors still consider cell injury as the only
form Of the response of hypersensitive cells to the antigen.
At.. our laboratory this problem has been thoroughly analyzed.
We used two experimental approached, viz.:the determination of the
migration of hypersensitive guinea pig cells? in the presence of
graded doses of sPecific antigen on the one hand /11/, and the
of the dynamics of migration at various time intervals. on
.the other hand /12/0 The experiments were performed using our
standard technique of ,spleen fragment cultivation in liquid medium
with objective photographic recoding of the increasing size of -
several parallelly cultivated, fragments /10/. The results are
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t'Ap.vezuu mo k.djuvid, a-ove *
the growth in experimental conditions and that in control culti-
vations.
In the first series of experiments with normal and hyper-
sensitive spleen fragments graded doses of antigen /PPD tuber-
culin ranging between 50 gamma to 0,02 gamma and of Old Tuber-
culin from 1 2 100 to 12 250 000/ were used. Higher antigen doses
usually used by Most authors studying similar problems regularly
result in an inhibition of the migration of hypersensitive cells.
The relatiOnship between the quantity of antigen used and the
:degree of migration inhibition is statistically significant. On
usingvery mall antigen doses i.e. PPD 0,5 to 0 1-gamma,
OT 1 2 2 000 to 1 2 50 000 We observe in some cases /in 23 out
of 56 experiments"with Sensitive cells/ a marked stimulation of
the migration; this result is statistically significant, Similar
stimulation was never observed in control cultivations using
nonsensitized cells or on using nonspecifically toxic antigen,
/purified Salmonella paratyphi B endotoxin/?-both in sensitized
as in normal cells.
In the se-cond series of experiments antigen doses used were
10 gamma PPD and 1 2 100 OT and migration dynamic was followed at
regular time intervals during the incubation of spleen frag-
ments. Cells of normal fragments with or without antigen And cells
of fragmentS from sensitized animals without antigen migrate rela-
tively evenly thoughout the experiment, On the other hand in
hypersensitive cells incubated with antigen a stimulation of
the migration activity is regularly observed in the first hours,
as Compared with the control groups, while later cells migration
is slowed down or ceases altogether, thus leading to the final
result observed after the end of the experiment as the inhibition of
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ration stimulation in the first hours of the experiment as well as t
the final inhibition are statistically significant. In control
experiments the effect of purified endotoxin was studied which in
low doses, causes a moderate stimulation of the migration activity
for the duration of the whole experiment while in higher doses
it leads to .a permanent inhibition from the beginning up to the
end of the experiment, both in normal and in sensitized cells.
The above mentioned results as well as the quoted experi-
ments of other authors permit to conclude that the response of
cells from a hypersensitive organism to sPecific antigen can appear
not only in the form of an inhibition, but also of a stimulation
of their activity. The result obviously depends on the conditions
of the experiment, antigen dose, degree of hypersensitivity, etc.
On the basis of single literary data as well as of our own pre-
,
liminary experiments we believe that the basis of the observed
reactions of hypersensitive cells incubated with antigen should be
seen in the changes of their metabolic activity.
The above results and conclusions must be brough into connect-
ion with the previously discussed hypothesis that a small per
of hypersensitive cells react specifically with antigen
and influence /probably as a result of the release of some me-
diator or mediators/ the behaviour of the other principally non-
sensitized cells. In other words, We have to admit that the
reaction of the cellular population to the proceeding reaction
of delayed type hypersensitivity can manifest itself by a stimulat-
ion of the function of these cells, probably as a consequence of
their increqsed metabolic activity.
This hypothetical conclusion is the very reason for dis-
cussing the results of the study-of delayed type hypersensitivity
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dUllallUV.Ky -
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at the Symposium on the Mechanism of Antibody Formation. The
exact relationship between delayed type hypersensitivity reactions
? and the mechanism of antibody formation is not known yot; 'there is,
at least, one fact we must keep in mind, i.e0 that in immunized
? animals usually both mechanisms exist simultaneously. It is known
at the same time that the process of antibody formation in vitro
?/especially the secondary response/ is accompanied by an increased
activity of the cellular suspension present which manifests itself
e.g0 as a stimulation of DNA synthesis /3,4/. We might perhaps
consider the possibility that in the metabolic changes observed at
the beginning of antibody formation in vitro participates the
simultaneously proceeding reaction of delayed hypersensitivity,
We could even presume that delayed type hypersensitivity reaction
connected with the general metabolic stimulation of the cell
population constitute the physiological basis for antibody format-
ion; all this is naturally merely a hypothesis.
The main interest of our studies lies exclusively in the
field of delayed type hypersensitivity. We think though that some of
the reported results can be interesting also for those studying the
mechanism of antibody formation. ?
References
1. David, J,R., Al-Askari, S., Lawrence H.S., and Thomas, L
Abstract Feder.Proc. 222 618 /1963/.
-2. Dittmar, C. and Sixel, J.t Beitr.Klin.Tuberk. 112:483, /1954/.
3. ? Dutton, R.W. and Eady, J.D.: Nature /London/ 194: 93 /1962/.
4, Dutton,:R.W. and Eady, J.D.: Immunology 7: 40 /1964/, and
Dutton, R.W', and Bulman, Ii N0: Immunology 54 /1964/.
50 Johanovs, J.: Lectures presented at Small Meeting EAA,
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r -
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?
JohanovskIr at al. - 7
Prague 1962, 5th European congress of allergy, Base 1962 and
Symposium on Delayed type hypersensitivty, Davos 1964.
Juhacz-Sch?er, A.: Zeitschr.f.Immunitatsforsch. 56: 377
/1928/.
7. Marshall, W.H. and Roberts, K.B.: Lancet 1: 773 /1963/.
8. Pearmain, G., Lycette, R.R. and Fitzgerald, P.H.: Lancet 1:
637 /1963/.
9. Pincus, W.B., Sokolic, I.H.,and Readier,
/1963/ and J.Allergy 35: 117 /1964/.
10. ?,'vejcar, J. and Johanovskfr, J.: Zeitschr.f.ImmunitUtsforsch.
122: 398, 420 and 438 /1962/.
11. Nrejcar, J. and Johanovsky', J.: to be published; abstract
in Proc. 5th European congress of allergy, Basel 1963,
p.375.
12. Irejcar, J. and Johanovskfr, Jet Folia Microbiol. 8: 245
/1963/ and Zeitschr.f.Immunitilts u.Allergieforsch. /in press/,
13, vejcar, J. and Johanovsk, J.: to be published; abstract
in Proc. 5th European congress of allergy, Basel 1963,
pp.248 and 375.
14. Waksman, B.H.: AmeRev.Tuberc. 68: 746 /1953/,
15. Waksman, B.H. and Matoltsy, M.: J.Immunol. 81: 222 /1958/.,
15. Wal]raff, E.B.: Proc.Soc.exp.Biol.Med, 113: 650 /1963/.
;Allergy
34: 337
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? ,
Intiunological Competence of Different ,Stas of :the '"LYthp`hei'd'
: 7
KamarSTtoVa.%,-
?
?
tlr;taP f, Microbiology; ',Czech,?VroVale A0 a 4erv 'of -s-cl.pail.oe'ai
,prp?51.1e, Cze'dhpsichr,akid
-.1Ontog,e-ifet,io'sche'Mb?.,Of :??-the devefdpinen,t ?Vr'mapmal.:.?1ym- .i:)..h.' ola, - ?
. , . . .. .
., , ... ..
.. ,. ... -... ,
,,..,
.,
- ?
cells ? yi,h.tchi 6/..e the6.-.6.ri-i,e,sw .
7- indw. r-,,;S?garded- t.o. be probably ?-,?'.a.c.Toll-fo*s.:, .:r154-1'irOt? ',1ymphotcytes-
. . , ?
? ''r 4:a.-. ,"
' eVel op' from' ?tha. eip.ithelia1 .ba.se of ' the ' thYPIHas :an?,a1.0go-i:i0...'? .
. ,
-, s. ?....- ? ? ? ? -? ?? -, r t, ..-. ', -?-??? ..., , ,'-? -.i,'' .'- ..- ? ' v ? , ?'" '
, o.rgank???-dnder :-the' humoral -influence of :,the.-- ineSenchyme.../2.; "1-9,
. . .. -
; These: iyinpllocyte:,:i.-1;seTriiii?ate?-,..1_T-ito,, the ;?cis'zketi.l.atioxi ,oalici --'s-eroori0?ary "..
,
rl.ymphatiotisues they- are either ./
. - ?
. , , . ?., ?
the stimulus;'-i:Or, the .prol4er,a,?'tion
,lyn:iphodytOpoiesis: /21/: or themselves" becOme111.4-r
any case; at the ?.erid, Of. theirieon-atal....periOd ,there ??
Is:: a ?,stii3ply" of small lymphocyte's at th perhery and in the*.
?...
, ? . ?
. .
"secondary-lymphatie organs Small lympho-9;t4 beT.6...4r6 caliab1e.
? .-. ? -
. ,
of transition?to1?.arge..1ymphqi4ii:/46:'.:iti* .-a60
. ? t ?
,
develbp. also 'from re serve"rof-_priniitives CelIS 4haraOter
. .
7 , of reticulum. cells/ in -which lymphocytopoiesis ?ban. ?aiso. -be
? .atithUlate.d by ,the humoral- effect of the- thyMio atr5Ma7,12,
The lymphocytopoiesis enhanaing:functiOn of the thymic
?
stroma :and. the medium of the ? -:thymuiseem?s'i to' be contradictory'
? the immunological reactivity :0g.. th.e lymooid, oe11.0 A3/
. .
SMa.11', lymphocytes in the ? 6:irbulatiOn and in the?''se ondaVi ' ? ?
ortar.40 like most lymphoid elL, hwe th'6 tjoo. of difrfe'rent-,
. ?
iqtion and modulation, into ' ,d12.0 pPss..042
STAT
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;3,? . - ?-rt ?
?
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, "- Holub et al. - 2
. (...
-
, immunological,competence /6/.,
? -In this work we have attempted to comparethe immunological
,
,
--0ftlpetence of lymphoid cells on their route from the thymus via'
_
: condarry lymphhtic.tissue.to the lymph and during their
,
. ,
-fOimation from large and small lymphocytes to histiocytes.
- e
Material and Methods
?
SO'urce of cells: All cells wore obtained from young
? ChincilIa rabbits. Thymus cells and 'mesenteric or media-
,stinal.' lymph:node cells *',7ere isolated from the organs by teastn,t?.
4 41.' ? -
thto 1.1.9n1; s or Earle 's solution. Lymph cells were obtained -by
,
, .dire'ct puncture of the cisterna chyli of donors immediately
after 'they had. been sacrificed and were resuspended in Hank's. ?
, . . .
-
.1 `? Oarle s solution with heparin, Histiocytes were Washed out
, the 'lungs in Hank's or Earle' s solution according to the' ?
. method of Myrwick et al. /1 4/. .
. ?
I ? .
" 2. Fractfonation of cell suspensions, The lymph node thfmus
and'-ilyriph cell suspensions were, separated tnto a fraction with
, .
..a ?completerpredominance of small lymphocytes an fractions with
77
s?
?
a higher: content of larger cells /other then small lymphocytes/
?
by;frgradient- 6entrifugation on layers of sucrose at 25, 30 and
3-0,oneentration 'in Hank's or Earle' s solution with 205 auto- ?
? ,
?,..
irabbit serum. Centrifugation was continued for 6 - 9 min.,
;
1, at - 30 g, and someticmes repeated, particularly in nodes. '
lifter- centrifuging, the cells were 'washed twice with i's' ?
or 'Earle' solutiOn, resuspended in the 'same mediuM an? d their
: ? ? ?
absolute number ?and differential count. 'determined.
' ,7?3. Antigen. Wi,th 'the exception of the control samples, BSA
,
? added to the\ cell suspension in amounts of 0,01 17-,
? , per .10-20x1ilijo cells.
4 1.
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,Holub et a1 -
I
4. Cultivatiori. Suspensions of'10-100.million cells vire
placed'in diffusion chambers of 0.8 mi?.vofume, vith''filters
c2 Czech production CTUFS, 0.1 - 0,3 11.1 porosity, exception71-.
Jy HUFS 0.3 - 0c5 Al porosity; in these filters the-great
nao-
tyof pores are smaller than 0.45 /u and'asdistinct,from the
filters .Millipore FA the7 were found,to be impermeable for cell
4
ard' in most cases with Ilembranfilter-Geseilschaft Gottingen
filters with a porosity of 0.15 0.275 ,u, Careful sealed
/
chambers were introduced into the peritoneal cavity ?of 3-6
Chinchilla rabbits treated with antibiotics. 1 - 3 chambers
. ?
. I
were placed in each infant rabbit. The rabbit t are not them-
.
selves capable of an antibody reaction to the amount of antig,-n
used The chambers were removed at, various time intervals. from
7 -.13 days, exceptionally after. longer cultivation
5.' Morphological analysis. Smears of the initial. ?susper.
and:smears and.imprinzs of the inner durface?oi.the chambe-r
filter after cultivation Tore fixe vet with Carnoy and ttined
rith methylene green-pyronine Y and also examined without t,..nat-
-.lent in phase contrast n SOMB cutivatioas filten imprints
.ere also ,rapidly dried, fixed with ethanol and stained by ti-ie
one-Stage method for antigen and the :two-stage method for .
.?ntibody using conugates of anti-BSA serum with florescein-
sothiocyanate apcording to a modification'of the method of
1 ?
:arshall'et al, /11/. DifferentlaLcounts Were made from
10,000 cells of the initial suspension and fro:D.1000-5000 cells
after cultivatlon.
6, Serological determinations: The fluid from the chambers
was used to determine the anti-BSA antibbdy.,COntent by the met*.ro-1
of passive microhaemagglutination with -anned, and. benzidinO-tro atr:'
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, .
tol'ub
sheep-erythrocytes,
. _
? 7, Types of 'experiMents. Antibody production and the ?number
. ? -
,
antiboay-icontairling, cells in the different .cell.-ivapensiOns
were -'determined after cultive.tiOn 'in the -separate chambers accord-
..
,
to the Method :described previbusly:/5/.
r -
?
? I - .
,The effedt of thymus stroma on the immun,o1ogida3, properties
cultivated lymphoid cells 'as -determined in twOTurther
?
modified cultivation methods: a. In Chambers' ,where thmus
?
StAme., t?obtainecl.' by .washing out the snTall cells trom. 6. small
?
fragment, of the' organ /2 x 2, mm./; ,'Was cultivated for',12:: and
da:ys-.Chambers filled _with Earle'._s-"solutiOn were'r:ple.6ed ?into
the recipients at the same, time;:*After1.12 and 4 d.ay 11 the
? . r , . d?
? . ?
chambers were taken. out and the fluid inside_ replaced. :throa
_ ,
? ? .
hole in the wan_ of the plexiglass "-ring with asuspensOnot_
lymphoifi 'cells freshly 3s olated. from lymph- nodes ? or lynpb with
antigen /BSA/.
Double chambers were made of two rings of TlexiglaSs
and closed by three filters, the centre' One /of .0.26 - 0.4- ,
'
?
po-rosity/-being . common to both asseTribled Chamberq. 'The muter. fii,t;.j
,
.$
_
ters had a porosity of 0.1.54 0.275 Fraentof thylauS
? :
/3 - 6 gr.iaginent '2 x 2 mm/ weie. enclobed;i..riione .thib.assembIed
-
,
ohambei-s-s:;-7'or asa tontrol, -.-fiagmen.?-ba
?
of lymph nodes. In the second Ofthe...asSembled.chambeSiaymphoid-
Cells. were ,; cultivated together with.:ant'igena/BAA/?,-,..?
" The ?total ,number of " chambers_ used in the: efipe,p.iments
?
be ,seen, from the tables, , ReCipients from one .litter ' were always
?
used for the i'cultivation of .differient typos of cells,'so that the
influence 'of indiviclqal factors of the recipient . On the':cell
?
,-4
suspensions should be minimal. In'sbme"exPerimentsyth`e bhan,iberb.
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?- ? ` r.. A
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lol- ? ? "
At ? 5) ?
,
? . ?
intervap:s;..
- ?
Trent. tabled 1 aria 2.. It is seen 'that a1 cu1tres
? ? A 'A
?
" t:aining more than ?10?,..? 10 lymph Or, lyMPh?.nOde-"Cells
r ? ? ? ",4 ? 1.. A . ?
4:. e
ant1.100#6V Inside the '.chamberS-1.ii
?
?
? ?
?
suspensions : of pure small lvtipl.).-6cytes froti th'ese "soOr.ces- are'
?, 4
,
somewhafi inferior in this respect. to suspans.ionaich,
,
? 'larger cells."... The production is both number 'and.,:ti?ITIE4 4ePeri--;
??, dent; :higher numbers' of Cells n starting suispensis ih,,mbs
? ? ? " ' ? -1= '
cases giVe -higher 'titres', of?'antioodies;r, , o
? . . ? . ? . . _
? ? ?
.? ,cultivation the production is nd1iib1e, the highei.tres!.?
'appear between the 8th and? 10th .day ? of 011)..tiya.tion
,
-titAnt 11:istiocytes.,/table ? contributP:'?
to ?antibody' formation?, because 'the: ? admix-1:3d aMoxint': -17friph-Otct;
cells "gave demonstrable antil:CO'die'sif''clil,ti?vAed'xiibne;', ?
,. ? ? , - ? T? ,. ":. ' :i".,?\:,
- .. ? . , _, ,
, the other hand, thymus , cells /table 3/ 0.47,:nof-AP.re;'-p.ily r -
relaibie;.titrea with the exception Df ?SePa,rateCt'smaili;thyrriUSY'
? ? -?,-
?
?
lymphocytes,' Whiah.preduce demonstrable 11:tr;a`? MiOre' than:60%
?
Of cultivations. Their' production. `1-6" agaiii '03716 aiicl'p'Prribpr'
?
dependent, In comparison with small lymphocytes from a4C'andary.
? ?
lymphoid tissues their production, is .,lower and a 'little,delaYed..
?
.? ^. ? - ? , . ? -
The aaMe picture is prOvided-bx? the ? *count of?antibodY'?''
cntainihg c.,13:s traced by the .inuriunpflgOrescent Metho4,7FigSaf.
? .
. , ? ?
?1.??5?-?2%?of Cells from lymph node and 'iymphare :dtained Opegifi:-
:c-alq..y on the 9th' day of cultivation, thymus am'al;lymPho'cyt.es ?
. are ? a. littlp inferior and thymus .cel-1:si,tsPenaldns incJgd,ing
?
? ? ? -,. ??
larger cells range .from Zero to -0.,!7%4 ?
4
lymph node! and lymph oultiVationo- Pia...sitia o? irtiniatir'6
ff p
plasma cells could be identified ,,among the antibodi;',COntai.ning <
? ,
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?.?