- Email: [email protected]
c mouse myeloma protein S117
DEVELOPMENTAL AND COM~PARATIVE IM@fUNOLOGY, Vol. 4, pp. 245-254, 1980. 014S-30SX/80/02024S-I0502.00/0 Printed in the U.S.A. Copyright (c) 1980 Pergamon Press Ltd. All rights reserved.
ANTI-IDIOTYPIC IgM ANTIBODIES PRODUCED IN CARPS WITH BALB/c MOUSE MYELOMA PROTEIN $117 FRANK EMMRICH o ROLAND F. RICHTER + and HERWART AMBROSIUS + °Staatliches Institut fEr Immunpr~parate und N~hrmedien, Berlin; +Section of Biosciences, Laborstory of Immunobiology, Karl-Marx-Universit~t, Leipzig, GDR.
ABSTRACT
Carps are able to synthesize xenogeneic anti-idiotypic antibodies against a mouse myeloma protein ($117) with specificity for N-acetyl-D-glucosamine. The properties of carp anti-idiotype (IgM) are compared to snti-idiotypic antisera from guinea pig (mainly IgG) elicited against the same idiotype. By radioprecipitin inhibition tests and passive hemagglutination inhibition tests, the specificity of carp and guinea pig snti-idiotypic antibodies is closely related. Furthermore, inhibition experiments with amino sugars suggest that at least some determinants localized in the antigen combining site of the $117 antibody are recognized by both carp and guinea pig anti-idiotypes. These results should contribute to the pkylogenetic aspect of idiotype anti-idiotype interaction and possibly also of idiotype regulation. INTRODUCTION
Xenogeneic anti-idiotypic antibodies are demonstrable by immunizing pkvlogeneticslly, highly developed mammals (I 3). However it is not as yet clear if anti-idiotypic antibodies sre produced also iD lower vertebrstes, for example in fishes, against mammalian idiotypes. The present paper describes the production of snti-idiotypic antibodies in such a pkylogenetically distant system. Carps snd guinea pigs were immunized with $117 myeloma protein from BALB/c mice (IgA/K) for comparison. $117 myeloma tumour growing in BALB/c mice has antibody activity against group A streptococcal carbokvdrate (4) and N-acetyl-D-glucosamine, the terminal in~mJnodeterminsnt group of the poLysaccharide. This opens the possibility of proving the specificity of anti-idiotypic sara by inhibition experiments using the specific hapten N-acetyl-D-glucosamine. S117-binding curves
246
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Vol. 4, No. 2
of anti-idiotypic antisera from guinea pigs (mainly IgG antibodies) and carps (exclusively tetrameric IgM antibodies) were compared and found to be very similar. MATERIALS and METHODS Animals. BALB/c mice were obtained from Sektion Tierproduktion, Versuchsststion Probstheida, KMU Leipzig. Mice bearing $117 plssmocytoms were kindly provided by Dr. Klaus Eichm~nn, Deutsches Krebsforschungszentrum Heidelberg, and propagated in our laboratory. For production of sntisera, colony bred guinea pigs were used and carps (1000-2000 g, maintained st 22°-24°C) from VEB Binnenfischerei Wermsdorf. Preparation of the idiotype. $117 idiotype was prepared by affinity chromatography of serum from m~eloms bearing mice on the specific amino sugar N-acetyl-D-glucosamine conjugated to Sepharose 4 B. p-nitrophenyl-~-N-scet¥1glucosaminide (Sigme Chemical Co.) was catalytically reduced to p-aminophenyl-6-N-acetylglucosaminide (5) and conjugated to CNBractivated Sepharose 4 B (6). $117 mveloms protein was eluted after specific binding onto the hapten-conjugated immunosorbent by 0.3 M N-acetylglucosamine and then dialyzed against PBS. The eluted $117 m~eloms protein was pure as shown by immunoelectrophoretical analysis. Antisera. Anti-idiotypic antiserum in IgG tolerant guinea pigs was prepared as previously described by simultaneous injection of 5 mg mouse IgG (i.v.) and 100/ug $117 myelonm protein with CFA (Difco Labs.) into the fo6tpads (7). Carps were immunized i.p. with 0.5 mg $117 m~eloma protein in 0.5 ml PBS, pH 7,4 emulsified 1:1 in CFA. After three months I mg $117 without adjuvant was applied i.p. and 14 days later carps were bled by cardiac puncture. Antiserum to carp immunoglobulin was prepared in rabbits as previously described (8). Antiserum to guinea pig immunoglobulin was obtained from Stastliches Institut ft~r Immunpr~parate und N~hrmedien, Berlin. . Mouse IgG was prepared according to LEVY and For absorption experiments mouse IgG and TEPC-15 (a generous gift from Dr. Humberto Cosenza) were conjugated to CNBr-activated Sepharose 4 B according to CUATRECASAS (10). Guinea pig and carp aid were absorbed first with mouse IgG Sephsrose and then with TEPC-15 Sepharose. To obtain a highly specific snti-idiotypic carp antiserum without significant loss of activity by unspecific absorption, different absorption procedures were tested. A procedure was selected using I mg mouse IgG and 2 mg TEPC-15 per ml anti-St17 serum of carp.
~
Abbreviations used in this paper: sld - snti-idiotype, PBS - phosphate buffered saline, CNBr - cysnogene bromide, CFA complete Freund's adjuvant, SRBC - sheep red blood cells, $117 - $117 n~eloms protein from BALB/c mice ~IgA/K), TEPC-15 - mveloms protein from BALB/c mice (IgATK). -
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Inhibition of idiotype-anti-idiotype-binding radioimmunoassa¥. $117 myeloma protein (idiotype) was radiola~eled with 12~-I using the chloramine-T methed (11). The specific activity after iodinstion was 5.4xi06 cpm/11ug $117. For quantitative inhibition tests, standard dilutiors of snti-idiotyoio sntiser8 were chosen giving 70-85% S117-binding (borate buffer pH 8.4; 0.2 % Tween 20; 1 % bovine serum albumin). To 501ul aid standard dilution increasing amounts of inhibitor'were added in 501ul assay-buffer containing equal amounts of preimmune g~inea pig or carp serum. After 30 min, 12 ng of 125-I-$117 i~ 501ul assay-bsffer were added and incubated for I h at 37 C arid 3 h st 4 C. Then as precipitating antibody 501ul goat anti-guinea pigf-globulin or rabbit snti-ca~p'immunoglobulin were sdde'd and incubated overnight at 4 C. The amount of bound 125-I-$117 was measured by counting radioactivity in the precipitates (100/ul) and supernatants (1001ul). Inhibition of passive hemaKglutination. Sheep erythrocytes were coupled wlth $117 myeloma protein (SOOpug per 10tul packed red cells) using bis-diazotized benzldine (12)4 Microtiter plates were used and 251uI of antiserum dilution, 201ul of 1 % standard horse serum Gilution (subsequent inhibitor~ volume) and 251ul of 0.4 % coupled erythrocyte suspension were pipetted into each cavity. For inhibition te~ts antisera and inhibitors were preincubsted 30 min at 37 C (13). RESULTS Precipitating antibodies were found in sll carps 14 days after the second injection of $117 myeloms protein. A further injection had no marked influence on the titer of precipitating antibodies. Therefore, the following experiments were done with antisers obtained after the second antigen injection. Anti-St17 antisera from carps were subsequently absorbed with another BALB/c myeloms protein (TEPC-15). In preliminary experiments an absorption procedure was selected using I mg mouse IgG and subsequently 2 mg TEPC-15 per ml anti-St17 serum, aid antisera from carps(IgM) absorbed in this way showed specificity like IgG aid antibodies from tolerant guinea pigs absorbed with mouse IgG and TEPC-15 (FIG. 2). Precipitation curves of absorbed and unabsorbed aid antiserum from carp compared to aid antiserum from guinea pig are shown in figure I. In figure 2 inhibition curves of carp and guinea pig aid are compared. The homologous idiotype $117, another BALB/c idiotype (TEPC-15) and normal mouse IgG were used as inhibitors. Very similar inhibition curves could be obtained with anti-idiotypic antibodies from the 2 species. Idiotype-binding was inhibited by different amino sugars for further characterization of the aid (PIG.3). Both guinea pig aid and also carp aid could be inhibited by the amino sugar N-acetyl-glucosamine. With amino sugar concentrations higher than 0.3 M unspecific inhibition could be observed also by N-acetyl-galactosamine.
248
ANTI-IDIOTYPIC
IgM IN CARPS
Vol. 4, No. 2
100"
.=_ 50 "ID C I
u
10 j,
?
\ 10 10'
\
\
" ~ a r pcarp51dm 5ld°~~ carp 51d~ % ~ , 10'
aidu
1~ 10' 10' dilut~n of antiserum
i0c
FIG. I
Absorbed versus unabsorbed anti-St17 sntisera (aid) from csrp end guinea pig added in dilutions to 1-125-$117 in the indirect radioprecipitin assay, gPu aid from guinea pig, unabsorbed; gPa aid from guinea pig, absorbed; carPu aid from carp, unsbsorbed; carps I aid from csrp, absorbed only with TEPC-15; csrPa 2 sId from csrp, absorbed with mouse IgG snd TEPC-15
Vol. 4, No. 2
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IgM IN CARPS
249
® ~" rnouse IgG c~50 t-
.c I
i--I
10
® m ~ . _ _ _ .TEPC- 15 - ~ mouse IgG 5O
_-,, =
10
ATEPC-15 -mouse IgG
117 0,001
0,01
0,I
1 10 .z~g Inhibitor
FIG. 2
Inhibition of the indirect precipitin resction between rsdiolsbeled $117 end snti-S117 from s) guines pig gPs (diluted 1:300) end b) carp (carps I diluted 1:50, open symbols; cerps 2 diluted 1:25, filled symB61s). As inhibitors $117 snd TEPC-~5 end pooled mouse IgG were sdded in dilution. Gost antiguinee pig immunoglobulio (1:8) and rsbbit snti-carp immunoglobulin (undiluted) were sdded es precipiteting sntisere.
250
ANTI-IDIOTYPIC
IgM IN CARPS
Vol.
4, No. 2
Inhibitors [IVr] --
--
V/////////////////.'~"
~ P/1//////////////. !
NAcGlu
o.,,tt 0,13 0,25
.
"-I"-b "t"
NAcGlu
:~
0,06 Glu 0,13 0,25
:J
+ t"
Glu
20 30 40 0 50 60 70 80 %I"tbinding using carp 51d~_ %I1=~-binding using guinea pig 61d~
0
PIQ. 3
Inhibition of the indirect precipitin assay between radiolabeled $117 and anti-S117 from carp and guinea pig (for details see description of Pig. I, 2). The amino sugars N-ecety1-D-glucossmine (NAcGIu), D-glucosamine (Glu), N-acetv1-D-galactosamine (NAcGal) and D-galactosamine (Gal) were used in dilution as inhibitors. Specificity of the carp aid could also be demonstrated in a hemsgglutination inhibition assay using S117-coated SRBC (Table I). TABLE I Specificity of S117-binding demonstrated by inhibition of passive hemsgglutination of S117-coated SRBC and aid antisere from carp and guinea pig. aid antisera °
inhibit°rs+ (/ug/ml) $117
from carp 1:512 diluted from guinea pig 1:128 diluted
TEPC-15
mouse IgG
=
0,05
> 1000
>1860
=
0,1
> 1000
>1860
°Titer of the antisera were four times the concentration of the passive hemagglutination end poiDt +complete inhibition
Vol. 4, No. 2
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251
DISCUSSION
If the network theory of Jerne is a common principle in immunoregulation, we should assume that lower vertebrates are also able to produce idiotypes and anti-idiotypic antibodies. Indeed in sn sllogeneic system using antiDNP antibodies of carps, sllogeneic anti-idiotypic antibodies could be elicited (14). Xenogeneic anti-idiotypic antibodies should be expected only in snimsls whose variable region gene repertoire is large enough to recognize the little antigenic differences which characterize the idiotype. All mammals tested, as yet, developed anti-idiotypic antibodies against idiotypes of other msm,~ls. Allogeneic as well as xenogeneic intrs-msmm~lisn snti-idiotypic antibodies may detect the same determinants closely related to the antigenbinding site (15, 16), furthermore, allogeneic (mouse to mouse) and xenogeneic (guinea pig to mouse) anti-idiotypic antibodies have similar cross-reaction patterns in radioprecipitin tests (I). In the present stu&y it was shown that pkvlogenetically very different xenogeneic snti-idiotypic antisera give similar inhibition curves of idiotypebinding in radioprecipitin tests and also similar specificity in passive hemagglutinstion inhibition tests. In the latter the carp aid antiserum was diluted to a greater extent than guinea pig aid antiserum possibly because of the multivalence of tetrsmeric carp IgM. Both carp aid and guinea pig aid share in the recognition of idiotypic determinants belonging to the antigen combining site as demonstrated by inhibition curves with different sugars. Anti-idiotypic specificity in fishes over a great pk¥1ogenetic distance could be demonstrated not only with a mouse idiotype, but also against s human myeloma protein (13). Binding of carp aid to the homologous myeloms protein could not be inhibited by five other human myeloma proteins. Because fish can recognize idiotypic determinants of msmmAls, it can be assumed that the V gene repertoire in fishes is very large even though fishes are regarded as pkvlogeneticslly relatively primitive having emerged about 400 million years ago. Moreover, recognition of mammalian idiotypes is a rather sophisticated function which may be interpreted to mean that either the idiotype/anti-idiotype network is s common attribute of high stability in pkylogeny or thst modern bony fish have acquired some immunological sophistication with time. Possibly we shall find strongly conserved idiotypes common in fishes and mammals if we compsre idiotypic structures of germ line variable genes. Further experiments should answer the question: Do such pkylogeneticslly ancient antibodies of fishes mediate any regulation by idiotype-antiidiotype interaction in fishes as they seem to do in mammals?
252
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The answer seems important to us for two reasons: Firstly, because of the pkvlogenetic view of idiotype-anti-idiotype regulation and secondly, a more prectical point, because of the potential applications of carp IgM for studies of some general regulstor~ properties of anti-idiotypic antibodies of the IgM class. Carp IgM is free from contamination by other classes of immunoglobulins and shows msny functional similarities to msmm~lian IgM, with one important exception, carp IgM does not activate msmm~lisn complement.
ACKNOWLEDGMENTS: We wish to thank for kind support and ms~y gifts, K. Eichmsnn (Heidelberg), H. Cosenze (Basel), R. Mohr (K~ln), W. K~hler (Jena), D. H~dge and P. Nuhn (Leipzig).
REFERENCES I. EICH~ANN, K.: Idiotypic identity of antibodies to streptococcal carbokydrete in inbred mice. Eur. J.Immunol. 2, 301, 1972. 2. SLATER, R.J., WARD, S.M. and KUNKEL, H.G.: Immunological relationships among the myeloms proteins. J.Exp.~ed. 101, 85, 1955. 3. HOPPER, J.E. and NISONOFF, A.: Individual antigenic specificity of immunoglobulins. Adv. Immunol. 13, 58, 1971. 4. VICARI, G., SHER, A., COHN, M. and KABAT, E.: Immunochemical studies on a mouse mveloms protein with specificity for certain B-linked terminal residues of N-acetylD-glucossmine. Immunochemistry 7, 829, 1970. 5. KUHN, R. and HAAS, H.J.: Braunes Palladiumox~d-kvdratbsrium-sulfat f~r katelytische Kydrierungen. Angew.Chem. 67, 785, 1955. 6. WOFSY, L. and BURR, B.: The use of affinity chromstograpkv for the specific purification of antibodies and antigens. J.Immunol. 103, 380, 1969. 7. HENNEY, C.S. and ISHIZAKA, K.: A simplified procedure for the preparation of immunoglobulin-class-specific antisere. J.Immunol. 103, 56, 1969. 8. RICHTER, R., PRENZEL, E.-M., HADGE, D., KOPPERSCHLAGER, G., AMBROSIUS, H.: Strukturelle und immunchemische Untersuchungen am Immunglobulin des Karpfens (Cyprinus carpio L.). I. Analyse am Gesamtmolek~l. Acts biol.med. germ. 30, 735, 1973.
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9. LEVY, H.B. and SOBER, H.A.: A simple chromatographic method for preparation of gamma globulins. Proc. Soc.Exp. Biol.and Med. 103, 250, 1960. 10. CUATRECASAS, P.: Protein purification by affinity chromatography derivatizations of agarose and polyacrTlamide beads. J.Biol.Chem. 245, 3059, 1970. 11. HUNTER, W.M. and GREENWOOD, F.C.: Preparation of Iodine131 labelled human growth hormone. Nature (London) 194, 495, 1962. 12. STAVITSKY, A.B. and ARQUILLA, E.R.: Micromethods for the study of proteins and antibodies. III. Procedure and applications of hemsgglutination and hemsgglutination inhibition reactions with bis-diszotized benzidine and protein conjugated red blood cells. J.Immunol. 74, 306, 1955. 13. RICHTER, R.F. and AMBROSIUS, H.: Anti-idiotypic antibodies of IgM-type produced in carp (C~prinus csrpiq L.). Eur.J. Immunol. 9, 578, 1979. 14. ~ACHULLA, H.G.K., RICHTER, R.F. and AMBROSIUS, H.: Idiotype expression in carp (Cyprinus csr_~q L.). Poster Abstr. No. 4 ~n Europ. I m m u 6 o l . M e e t i n g ~ 8 Budapest, Ungarn. 15. BRIENT, B.W., HAIMOVICH, J. and NISONOFF, A.: Reaction of snti-idiotypic antibody with the hapten-binding site of a mTeloms protein. Proc.nat. Acad.Sci.U.S. 68, 3136, 1971. 16. SIRISINHA, S. and EISEN, H.: Autoimmune-like antibodies to the ligand-binding sites of mveloms protein. Proc. nat.Acad. Sci.U.S. 68, 3130, 1971.
ANTI-IDIOTYPIC IgM ANTIBODIES PRODUCED IN CARPS WITH BALB/c MOUSE MYELOMA PROTEIN $117 FRANK EMMRICH o ROLAND F. RICHTER + and HERWART AMBROSIUS + °Staatliches Institut fEr Immunpr~parate und N~hrmedien, Berlin; +Section of Biosciences, Laborstory of Immunobiology, Karl-Marx-Universit~t, Leipzig, GDR.
ABSTRACT
Carps are able to synthesize xenogeneic anti-idiotypic antibodies against a mouse myeloma protein ($117) with specificity for N-acetyl-D-glucosamine. The properties of carp anti-idiotype (IgM) are compared to snti-idiotypic antisera from guinea pig (mainly IgG) elicited against the same idiotype. By radioprecipitin inhibition tests and passive hemagglutination inhibition tests, the specificity of carp and guinea pig snti-idiotypic antibodies is closely related. Furthermore, inhibition experiments with amino sugars suggest that at least some determinants localized in the antigen combining site of the $117 antibody are recognized by both carp and guinea pig anti-idiotypes. These results should contribute to the pkylogenetic aspect of idiotype anti-idiotype interaction and possibly also of idiotype regulation. INTRODUCTION
Xenogeneic anti-idiotypic antibodies are demonstrable by immunizing pkvlogeneticslly, highly developed mammals (I 3). However it is not as yet clear if anti-idiotypic antibodies sre produced also iD lower vertebrstes, for example in fishes, against mammalian idiotypes. The present paper describes the production of snti-idiotypic antibodies in such a pkylogenetically distant system. Carps snd guinea pigs were immunized with $117 myeloma protein from BALB/c mice (IgA/K) for comparison. $117 myeloma tumour growing in BALB/c mice has antibody activity against group A streptococcal carbokvdrate (4) and N-acetyl-D-glucosamine, the terminal in~mJnodeterminsnt group of the poLysaccharide. This opens the possibility of proving the specificity of anti-idiotypic sara by inhibition experiments using the specific hapten N-acetyl-D-glucosamine. S117-binding curves
246
ANTI-IDIOTYPIC
IgM IN CARPS
Vol. 4, No. 2
of anti-idiotypic antisera from guinea pigs (mainly IgG antibodies) and carps (exclusively tetrameric IgM antibodies) were compared and found to be very similar. MATERIALS and METHODS Animals. BALB/c mice were obtained from Sektion Tierproduktion, Versuchsststion Probstheida, KMU Leipzig. Mice bearing $117 plssmocytoms were kindly provided by Dr. Klaus Eichm~nn, Deutsches Krebsforschungszentrum Heidelberg, and propagated in our laboratory. For production of sntisera, colony bred guinea pigs were used and carps (1000-2000 g, maintained st 22°-24°C) from VEB Binnenfischerei Wermsdorf. Preparation of the idiotype. $117 idiotype was prepared by affinity chromatography of serum from m~eloms bearing mice on the specific amino sugar N-acetyl-D-glucosamine conjugated to Sepharose 4 B. p-nitrophenyl-~-N-scet¥1glucosaminide (Sigme Chemical Co.) was catalytically reduced to p-aminophenyl-6-N-acetylglucosaminide (5) and conjugated to CNBractivated Sepharose 4 B (6). $117 mveloms protein was eluted after specific binding onto the hapten-conjugated immunosorbent by 0.3 M N-acetylglucosamine and then dialyzed against PBS. The eluted $117 m~eloms protein was pure as shown by immunoelectrophoretical analysis. Antisera. Anti-idiotypic antiserum in IgG tolerant guinea pigs was prepared as previously described by simultaneous injection of 5 mg mouse IgG (i.v.) and 100/ug $117 myelonm protein with CFA (Difco Labs.) into the fo6tpads (7). Carps were immunized i.p. with 0.5 mg $117 m~eloma protein in 0.5 ml PBS, pH 7,4 emulsified 1:1 in CFA. After three months I mg $117 without adjuvant was applied i.p. and 14 days later carps were bled by cardiac puncture. Antiserum to carp immunoglobulin was prepared in rabbits as previously described (8). Antiserum to guinea pig immunoglobulin was obtained from Stastliches Institut ft~r Immunpr~parate und N~hrmedien, Berlin. . Mouse IgG was prepared according to LEVY and For absorption experiments mouse IgG and TEPC-15 (a generous gift from Dr. Humberto Cosenza) were conjugated to CNBr-activated Sepharose 4 B according to CUATRECASAS (10). Guinea pig and carp aid were absorbed first with mouse IgG Sephsrose and then with TEPC-15 Sepharose. To obtain a highly specific snti-idiotypic carp antiserum without significant loss of activity by unspecific absorption, different absorption procedures were tested. A procedure was selected using I mg mouse IgG and 2 mg TEPC-15 per ml anti-St17 serum of carp.
~
Abbreviations used in this paper: sld - snti-idiotype, PBS - phosphate buffered saline, CNBr - cysnogene bromide, CFA complete Freund's adjuvant, SRBC - sheep red blood cells, $117 - $117 n~eloms protein from BALB/c mice ~IgA/K), TEPC-15 - mveloms protein from BALB/c mice (IgATK). -
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Inhibition of idiotype-anti-idiotype-binding radioimmunoassa¥. $117 myeloma protein (idiotype) was radiola~eled with 12~-I using the chloramine-T methed (11). The specific activity after iodinstion was 5.4xi06 cpm/11ug $117. For quantitative inhibition tests, standard dilutiors of snti-idiotyoio sntiser8 were chosen giving 70-85% S117-binding (borate buffer pH 8.4; 0.2 % Tween 20; 1 % bovine serum albumin). To 501ul aid standard dilution increasing amounts of inhibitor'were added in 501ul assay-buffer containing equal amounts of preimmune g~inea pig or carp serum. After 30 min, 12 ng of 125-I-$117 i~ 501ul assay-bsffer were added and incubated for I h at 37 C arid 3 h st 4 C. Then as precipitating antibody 501ul goat anti-guinea pigf-globulin or rabbit snti-ca~p'immunoglobulin were sdde'd and incubated overnight at 4 C. The amount of bound 125-I-$117 was measured by counting radioactivity in the precipitates (100/ul) and supernatants (1001ul). Inhibition of passive hemaKglutination. Sheep erythrocytes were coupled wlth $117 myeloma protein (SOOpug per 10tul packed red cells) using bis-diazotized benzldine (12)4 Microtiter plates were used and 251uI of antiserum dilution, 201ul of 1 % standard horse serum Gilution (subsequent inhibitor~ volume) and 251ul of 0.4 % coupled erythrocyte suspension were pipetted into each cavity. For inhibition te~ts antisera and inhibitors were preincubsted 30 min at 37 C (13). RESULTS Precipitating antibodies were found in sll carps 14 days after the second injection of $117 myeloms protein. A further injection had no marked influence on the titer of precipitating antibodies. Therefore, the following experiments were done with antisers obtained after the second antigen injection. Anti-St17 antisera from carps were subsequently absorbed with another BALB/c myeloms protein (TEPC-15). In preliminary experiments an absorption procedure was selected using I mg mouse IgG and subsequently 2 mg TEPC-15 per ml anti-St17 serum, aid antisera from carps(IgM) absorbed in this way showed specificity like IgG aid antibodies from tolerant guinea pigs absorbed with mouse IgG and TEPC-15 (FIG. 2). Precipitation curves of absorbed and unabsorbed aid antiserum from carp compared to aid antiserum from guinea pig are shown in figure I. In figure 2 inhibition curves of carp and guinea pig aid are compared. The homologous idiotype $117, another BALB/c idiotype (TEPC-15) and normal mouse IgG were used as inhibitors. Very similar inhibition curves could be obtained with anti-idiotypic antibodies from the 2 species. Idiotype-binding was inhibited by different amino sugars for further characterization of the aid (PIG.3). Both guinea pig aid and also carp aid could be inhibited by the amino sugar N-acetyl-glucosamine. With amino sugar concentrations higher than 0.3 M unspecific inhibition could be observed also by N-acetyl-galactosamine.
248
ANTI-IDIOTYPIC
IgM IN CARPS
Vol. 4, No. 2
100"
.=_ 50 "ID C I
u
10 j,
?
\ 10 10'
\
\
" ~ a r pcarp51dm 5ld°~~ carp 51d~ % ~ , 10'
aidu
1~ 10' 10' dilut~n of antiserum
i0c
FIG. I
Absorbed versus unabsorbed anti-St17 sntisera (aid) from csrp end guinea pig added in dilutions to 1-125-$117 in the indirect radioprecipitin assay, gPu aid from guinea pig, unabsorbed; gPa aid from guinea pig, absorbed; carPu aid from carp, unsbsorbed; carps I aid from csrp, absorbed only with TEPC-15; csrPa 2 sId from csrp, absorbed with mouse IgG snd TEPC-15
Vol. 4, No. 2
ANTI-IDIOTYPIC
IgM IN CARPS
249
® ~" rnouse IgG c~50 t-
.c I
i--I
10
® m ~ . _ _ _ .TEPC- 15 - ~ mouse IgG 5O
_-,, =
10
ATEPC-15 -mouse IgG
117 0,001
0,01
0,I
1 10 .z~g Inhibitor
FIG. 2
Inhibition of the indirect precipitin resction between rsdiolsbeled $117 end snti-S117 from s) guines pig gPs (diluted 1:300) end b) carp (carps I diluted 1:50, open symbols; cerps 2 diluted 1:25, filled symB61s). As inhibitors $117 snd TEPC-~5 end pooled mouse IgG were sdded in dilution. Gost antiguinee pig immunoglobulio (1:8) and rsbbit snti-carp immunoglobulin (undiluted) were sdded es precipiteting sntisere.
250
ANTI-IDIOTYPIC
IgM IN CARPS
Vol.
4, No. 2
Inhibitors [IVr] --
--
V/////////////////.'~"
~ P/1//////////////. !
NAcGlu
o.,,tt 0,13 0,25
.
"-I"-b "t"
NAcGlu
:~
0,06 Glu 0,13 0,25
:J
+ t"
Glu
20 30 40 0 50 60 70 80 %I"tbinding using carp 51d~_ %I1=~-binding using guinea pig 61d~
0
PIQ. 3
Inhibition of the indirect precipitin assay between radiolabeled $117 and anti-S117 from carp and guinea pig (for details see description of Pig. I, 2). The amino sugars N-ecety1-D-glucossmine (NAcGIu), D-glucosamine (Glu), N-acetv1-D-galactosamine (NAcGal) and D-galactosamine (Gal) were used in dilution as inhibitors. Specificity of the carp aid could also be demonstrated in a hemsgglutination inhibition assay using S117-coated SRBC (Table I). TABLE I Specificity of S117-binding demonstrated by inhibition of passive hemsgglutination of S117-coated SRBC and aid antisere from carp and guinea pig. aid antisera °
inhibit°rs+ (/ug/ml) $117
from carp 1:512 diluted from guinea pig 1:128 diluted
TEPC-15
mouse IgG
=
0,05
> 1000
>1860
=
0,1
> 1000
>1860
°Titer of the antisera were four times the concentration of the passive hemagglutination end poiDt +complete inhibition
Vol. 4, No. 2
ANTI-IDIOTYPIC
IgM IN CARPS
251
DISCUSSION
If the network theory of Jerne is a common principle in immunoregulation, we should assume that lower vertebrates are also able to produce idiotypes and anti-idiotypic antibodies. Indeed in sn sllogeneic system using antiDNP antibodies of carps, sllogeneic anti-idiotypic antibodies could be elicited (14). Xenogeneic anti-idiotypic antibodies should be expected only in snimsls whose variable region gene repertoire is large enough to recognize the little antigenic differences which characterize the idiotype. All mammals tested, as yet, developed anti-idiotypic antibodies against idiotypes of other msm,~ls. Allogeneic as well as xenogeneic intrs-msmm~lisn snti-idiotypic antibodies may detect the same determinants closely related to the antigenbinding site (15, 16), furthermore, allogeneic (mouse to mouse) and xenogeneic (guinea pig to mouse) anti-idiotypic antibodies have similar cross-reaction patterns in radioprecipitin tests (I). In the present stu&y it was shown that pkvlogenetically very different xenogeneic snti-idiotypic antisera give similar inhibition curves of idiotypebinding in radioprecipitin tests and also similar specificity in passive hemagglutinstion inhibition tests. In the latter the carp aid antiserum was diluted to a greater extent than guinea pig aid antiserum possibly because of the multivalence of tetrsmeric carp IgM. Both carp aid and guinea pig aid share in the recognition of idiotypic determinants belonging to the antigen combining site as demonstrated by inhibition curves with different sugars. Anti-idiotypic specificity in fishes over a great pk¥1ogenetic distance could be demonstrated not only with a mouse idiotype, but also against s human myeloma protein (13). Binding of carp aid to the homologous myeloms protein could not be inhibited by five other human myeloma proteins. Because fish can recognize idiotypic determinants of msmmAls, it can be assumed that the V gene repertoire in fishes is very large even though fishes are regarded as pkvlogeneticslly relatively primitive having emerged about 400 million years ago. Moreover, recognition of mammalian idiotypes is a rather sophisticated function which may be interpreted to mean that either the idiotype/anti-idiotype network is s common attribute of high stability in pkylogeny or thst modern bony fish have acquired some immunological sophistication with time. Possibly we shall find strongly conserved idiotypes common in fishes and mammals if we compsre idiotypic structures of germ line variable genes. Further experiments should answer the question: Do such pkylogeneticslly ancient antibodies of fishes mediate any regulation by idiotype-antiidiotype interaction in fishes as they seem to do in mammals?
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The answer seems important to us for two reasons: Firstly, because of the pkvlogenetic view of idiotype-anti-idiotype regulation and secondly, a more prectical point, because of the potential applications of carp IgM for studies of some general regulstor~ properties of anti-idiotypic antibodies of the IgM class. Carp IgM is free from contamination by other classes of immunoglobulins and shows msny functional similarities to msmm~lian IgM, with one important exception, carp IgM does not activate msmm~lisn complement.
ACKNOWLEDGMENTS: We wish to thank for kind support and ms~y gifts, K. Eichmsnn (Heidelberg), H. Cosenze (Basel), R. Mohr (K~ln), W. K~hler (Jena), D. H~dge and P. Nuhn (Leipzig).
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9. LEVY, H.B. and SOBER, H.A.: A simple chromatographic method for preparation of gamma globulins. Proc. Soc.Exp. Biol.and Med. 103, 250, 1960. 10. CUATRECASAS, P.: Protein purification by affinity chromatography derivatizations of agarose and polyacrTlamide beads. J.Biol.Chem. 245, 3059, 1970. 11. HUNTER, W.M. and GREENWOOD, F.C.: Preparation of Iodine131 labelled human growth hormone. Nature (London) 194, 495, 1962. 12. STAVITSKY, A.B. and ARQUILLA, E.R.: Micromethods for the study of proteins and antibodies. III. Procedure and applications of hemsgglutination and hemsgglutination inhibition reactions with bis-diszotized benzidine and protein conjugated red blood cells. J.Immunol. 74, 306, 1955. 13. RICHTER, R.F. and AMBROSIUS, H.: Anti-idiotypic antibodies of IgM-type produced in carp (C~prinus csrpiq L.). Eur.J. Immunol. 9, 578, 1979. 14. ~ACHULLA, H.G.K., RICHTER, R.F. and AMBROSIUS, H.: Idiotype expression in carp (Cyprinus csr_~q L.). Poster Abstr. No. 4 ~n Europ. I m m u 6 o l . M e e t i n g ~ 8 Budapest, Ungarn. 15. BRIENT, B.W., HAIMOVICH, J. and NISONOFF, A.: Reaction of snti-idiotypic antibody with the hapten-binding site of a mTeloms protein. Proc.nat. Acad.Sci.U.S. 68, 3136, 1971. 16. SIRISINHA, S. and EISEN, H.: Autoimmune-like antibodies to the ligand-binding sites of mveloms protein. Proc. nat.Acad. Sci.U.S. 68, 3130, 1971.