- Email: [email protected]
[9] Use of immunoblotting to detect idiotypic determinants on monoclonal antibodies
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[9]
meric IgM alone and in complex with IgG anti-Id, respectively (K. H. Roux, T. J. Elliott, and M. J. Glennie, unpublished data). In some instances, IgM monomers may be more appropriate targets for complexing with anti-Id antibody. To avoid misinterpretation, care must be taken to ensure that the IgM monomers derived from reduced and alkylated pentamers retain intact heavy-heavy and heavy-light interchain disulfide bonds (Fig. 9C). 7 As is dramatically shown in Fig. 9D, the noncovalent forces holding a partially reduced IgM monomeric subunit together in solution are insufficient to maintain proper configuration or in some instances to keep the two heavy-light halves of the molecules together following interaction with an IgG anti-Id antibody. Acknowledgments I would like to thank Drs. N. Greenspan, J. Davie, T. Elliott, and M. Glennie for use of unpublished material and T. Fellers for reviewing the manuscript. This work was supported by National Institutes of Health Research Grant AI16596.
[9] U s e o f I m m u n o b l o t t i n g to D e t e c t I d i o t y p i c D e t e r m i n a n t s on Monoclonal Antibodies B y C . P E T I T , M . E . SAURON, M . GILBERT, a n d J . T H ~ Z E
Introduction A technique for the transfer of proteins from gels to paper has been developed ("protein blotting"). 1,2Three steps are involved: (1) separation of proteins by gel electrophoresis in the presence of urea, ampholytes, or sodium dodecyl sulfate (SDS), (2) transfer of these proteins to diazobenzyloxymethylcellulose (DBM) paper I or to nitrocellulose filters by electrophoresis or diffusion, 2,3 and (3) detection of the adsorbed proteins by incubation of the filter with specific reagents. The proteins can be i H. A. Erlich, J. R. Levinson, S. N. Cohen, and H. D. Devitt, J. Biol. Chem. 254, 12240 (1979). 2 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). 3 B. Bowen, J. Steinberg, U. K. Laemmli, and H. Weintraub, Nucleic Acids Res. 1, 1
(1980). METHODS IN ENZYMOLOGY,VOL. 178
Copyright© 1989by AcademicPress, Inc. All rightsof rcproductinnin any form reserved.
[9]
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identified by the binding of specific antibodies 2 or by their ability to specifically bind ligands such as DNA, RNA, or histones. 3 This latter property indicates that proteins first separated on SDS gel and then transferred to nitrocellulose filters by diffusion have been at least partly renatured. In this chapter, we report on the utilization of the protein-blotting technique to study the idiotypic determinants expressed by an antipoly(Glu6°-Ala3°-Tyr1°) (GAT) monoclonal antibody (G5). Using polyclonal anti-idiotypic sera, two idiotypic specificities in particular have been studied on anti-GAT hybridoma products (HP): (I) the public idiotypic specificity (p. GAT) present in an identical form in all anti-GAT HP and all anti-GAT antibodies of every mouse strain tested 4 and (2) the highly conserved idiotypic specificity (h.c. GAT) also present on rat and guinea pig anti-GAT antibodies. 5 Thesetwo idiotypic specificities can be detected on blotted G5 molecules, previously run on a SDS-polyacrylamide gel. For this study, protein transfer was performed essentially according to the technique described by Bowen et al. 3 However, to favor the renaturation of idiotypic determinants of the blotted proteins and to minimize nonspecific binding, we have introduced some modifications of the method.
Materials
Monoclonal lmmunoglobulin, Antisera, and Purified Antibodies
Hybridoma cell line G5Bb2-2 was obtained by fusion of GAT-sensitized BALB/c spleen cells with myeloma cell line X-63 Ag8-NS-1. The corresponding HP (G5) (yl, r) was purified on GAT-Sepharose beads. 6 MOPC-21 (yl, K) protein was given by Dr. Juy. Rabbit anti-mouse Ig immune serum (R anti-M Ig) was given by T. Ternynck. Purified sheep anti-rabbit Ig antibodies free of cross-reactivity with mouse Ig were prepared as previously described 7 and labeled with horseradish peroxidase (PO) using a two-step procedure. 8 Normal BALB/c immunoglobulins were purified from normal BALB/c ascitic fluid as previously described. 9 4 G. Somm6, L. Leclercq, C. Petit, and J. Th6ze, Eur. J. Immunol. 11, 493 (1981). 5 G. Somm6 and J. Th6ze, Mol. lmmunol. 16, 1119 (1979). 6 L. Leclercq, J. C. Mazi6, G. Somm6, and J. Th6ze, Mol. lmmunol. 19, 1001 (1982). 7 C. Petit, J. C. Antoine, and S. Avram6as, lmmunochemistry 14, 479 (1977). s S. Avram6as and T. Ternynck, Immunochemistry 9, 1175 (1971). 9 j. Th~ze and G. Somm6, Fur. J. lmmunol. 9, 294 (1979).
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Anti-Idiotypic Sera The anti-idiotypic immune serum 715-7A4 was raised in a rabbit against BALB/c anti-GAT antibodies. The specificity and the characterization of this serum have been reported. 9 The serum 715-7A4 recognizes the p. GAT idiotypic specificity on the monoclonal BALB/c anti-GAT antibodies. 6 The anti-idiotypic immune serum 13-7A1 was raised in a rabbit against F.344 rat anti-GAT antibodies. The properties of this serum have been analyzed; the serum recognizes the h.c. GAT idiotypic specificity on monoclonal BALB/c anti-GAT antibodies. ~° Procedure
Gel Electrophoresis and Protein Transfer SDS-polyacrylamide gel electrophoresis was carried out as described by Laemmli. n Samples containing 10/~g of protein were diluted in "sample buffer" with or without 2-mercaptoethanol. Nonreduced samples were allowed to migrate on gels containing 7.5% acrylamide and 0.4% methylbisacrylamide. Reduced samples were separated on gels containing 15% acrylamide and 0.4% methylbisacrylamide. Immediately after electrophoresis, the SDS gel was shaken in transfer buffer (50 mM NaC1, 2 mM Tris-HCl, pH 7.4, 0.I mM phenylmethylsulfonyl fluoride) for 15 min at 20 °. The gel was then sandwiched between two sheets of nitrocellulose filters equilibrated in transfer buffer and two Whatman 3 MM filter papers and pressed together in a holder as described by Bowen et al. 3 The holder was immersed for 18 hr in 2 liter of transfer buffer under agitation at room temperature. After transfer, the nitrocellulose sheets were incubated for 30 min at 20°, with shaking, in phosphate-buffered saline (PBS: 10-z M phosphate buffer, pH 7.4, 0.15 M NaCI) containing 3% Nonidet P-40 (NP-40), then rinsed (3 times, 5 min each) in PBS. The nitrocellulose filters were finally incubated for 1 hr at 37° in saline buffer (0.9% NaCI, 10 mM Tris-HC1, pH 7.4) with 3% bovine serum albumin (BSA) to saturate the remaining protein binding sites of the nitrocellulose filters. This was followed by a brief washing in saline buffer containing 1%o Tween. The protein transfer was checked by staining of a strip of the nitrocellulose filters with Amido Schwarz 12(amido black) and staining of SDS gels with Coomassie blue. to C. Petit, M. Gilbert, G. Somm6, J. C. Mazi6, M. Dorf, and J. Th~ze, Mol. Irnmunol. 19, 1139 (1982). n U. K. Laemmli, Nature (London) 2,27, 680 (1970). 12 W. Schaffner and C. Weissmann, Anal. Biochem. 56, 502 (1973).
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Binding of Immune Reagents and Detection of Anti-PO Activity Strips cut from the nitrocellulose filters were incubated for 45 min in the presence of either rabbit anti-idiotypic serum, or R anti-M Ig serum, or normal rabbit serum (NRS). The anti-idiotypic sera were first diluted in NRS (v/v) and further diluted in PBS containing 1% Tween and 3% BSA. The R anti-IgM serum and the NRS were diluted in PBS, I% Tween, 3% BSA. The R anti-lgM serum was used at 1/200 dilution, the NRS at 1/20 dilution. All incubations were performed in small, heat-sealed plastic bags, so as to minimize the volume of reagents. Thereafter, the strips were rinsed (3 times, 10 min each in washing buffer (0.5 M NaCI, 10 mM Tris-HCl, pH 7.4, 1%o Tween) at 20° with shaking. PO-labeled sheep antirabbit Ig antibodies were then allowed to react with rabbit Ig, in PBS, 1% Tween, 3% BSA. After this last incubation, the strips of nitrocellulose were rinsed in the washing buffer. Strips were incubated for 5 min in the PO assay buffer (0.1 mM TrisHCI, pH 7.6), then placed in the same buffer containing 500/zg/ml of 3,3'diaminobenzidine (DAB) (Prolabo, Paris) and 1/zl/ml of H202 (30%). The enzymatic reaction was stopped at various times ranging from a few seconds to 5 min by immersion of the strips in water containing 0. I N HCI.
Detection of Conformational Idiotypic Specificities on a BALB/c Anti-GAT Antibody (GS) After migration on a SDS-polyacrylamide gel under nonreducing conditions and transfer by diffusion to a nitrocellulose filter, the monoclonal BALB/c anti-GAT antibody G5 was stained by the anti-idiotypic serum 715-7A4 (Fig. 1). Within 20 to 30 sec, an intense precipitate of DAB was formed when the anti-idiotypic serum was used at 1/20 dilution and the sheep anti-rabbit immunoglobulin antibodies at 40/~g/ml. Under the same experimental conditions, normal BALB/c immunoglobulins were only very weakly stained. Similarly, the MOPC-21 myeloma protein, which possesses a yl heavy chain and a r light chain like G5, was very faintly stained after incubation with serum 715-7A4. The ability of the anti-idiotypic serum 13-7A1 to specifically bind the G5 molecules after transfer was also tested. As shown in Fig. 1, after incubation with serum 13-7A1, intense staining of the G5 molecules was observed while very faint staining of normal BALB/c or MOPC-21 immunoglobulins was noted. This staining appeared in a few seconds when a 1/20 dilution of serum 13-7A1 was used with a concentration of anti-rabbit immunoglobulin antibodies of 40/xg/ml. The specificity of this idiotypic binding was checked. When G5 was incubated in presence of NRS, no staining of G5 by the sheep anti-rabbit immunoglobulins antibodies was observed (Fig. 1).
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FIG. 1. Detection of p. GAT and h.c. GAT idiotypic specificities on a monoclonal BALB/c anti-GAT antibody, G5. G5 (3'~, K), MOPC-21, and purified normal BALB/c immunoglobulin (NIg) were allowed to migrate on an SDS-polyacrylamide gel under nonreducing conditions. They were then blotted onto a nitrocellulose filter and incubated with 715-7A4, 13-7A1, or NRS. The enzymatic staining was stopped after 30 sec. T w o additional bands with apparent molecular weights slightly lower than G5 w e r e heavily stained b y s e r u m 715-7A4 and serum 13-7A1. These r e p r e s e n t e d v e r y minor c o m p o n e n t s as judged by Amido Schwarz (amido black) staining of the strips or C o o m a s s i e blue staining of the gels before blotting. T h e y m a y represent the products of a limited proteolysis or a n o n h o m o g e n e o u s denaturation by SDS, or heterogeneous glycosylation 13 of protein G5. The v e r y w e a k staining o f normal B A L B / c and MOPC-21 immunoglobulins b y serum 13-7A1 and 715-7A4 m a y be due to v e r y small amounts o f anti-isotypic antibodies in these sera. Optimal detection of the serum 715-7A4 and serum 13-7A1 bindings was obtained with the serum and antibody concentrations reported above. H o w e v e r , lowering the concentration o f b o t h reagents (up to 1/100 dilution for the immune serum and 10/zg/ml for the labeled antibodies) only slightly decreased the detection of the idiotypic determinants. 13R. Pitt-Rivers and F. S. A. Umpiom-Bato, Biochem. J. 109, 825 (1968).
[9]
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Comments
Some technical points have to be discussed. First, transfer by electrophoresis (for 1.5 hr, under 200 mA, in 192 mM glycine, 25 mM Tris buffer) was less efficient than transfer by diffusion. This was judged by Amido Schwartz staining of blotted G5 molecules and may be explained by the fact that nitrocellulose paper binds molecules moving under an electric field less efficiently. Second, an increase of the binding of the two antiidiotypic sera to the blotted G5 molecules was obtained by incubation of blotted proteins in NP-40 immediately after the transfer; this result can be attributed to the effect of NP-40 on the renaturation of proteins previously denatured by SDS as reported by Ames and Nikaido.14 Finally, the significant nonspecific binding to the nitrocellulose filters observed in preliminary experiments was prevented by addition of 1% Tween in the incubation and washing buffers. Similarly, washing with 0.5 M NaCI eliminates the nonspecific binding. As a general rule, both specific heavy and light chains are required for the expression of idiotypic determinants. However, in some cases, idiotypes have been detected on isolated chains. 15 We have attempted to detect the p. GAT and the h.c. GAT idiotypic specificities on H5 and L5 separated chains after protein transfer. We failed to reveal the p. GAT or the h.c. GAT specificities on H5 or L5 using the same experimental conditions permitting their detection on whole G5 molecules. Preincubation of the separated chains with NP-40 did not change the results. Thus, the association of H5 and L5 chains is necessary for the expression of the p. GAT and the h.c. GAT specificities. These results have been confirmed by Somm6 et al.16 They observed that isolated H5 and L5 chains do not inhibit the binding of serum 715-7A4 and serum 13-7A1 to radiolabeled G5 molecules. Thus, the results obtained by the protein-blotting technique and radioimmunoassay are in complete agreement, and demonstrate that p. GAT and h.c. GAT are conformational idiotypic specificities. The ability to detect conformational idiotypic determinants on molecules previously migrated on SDS gels, under nonreducing conditions, indicates that protein blotting followed by the treatment described above allows a satisfactory renaturation of blotted proteins.
14j. F. L. Ames and K. Nikaido, Biochemistry 15, 616 (1976). l~ A. Nisonoff, J. E. Hopper, and S. B. Spring, "The Antibody Molecule," p. 481. Academic Press, New York, 1975. 16 G. Somm6, J. Rocca-Serra, L. Leclercq, J. C. Mazi6, L L. Moreau, D. Moinier, M. Fougereau, and J. Th~ze, Mol. Immunol. 19, 1010 (1982).
IDIOTYPES, ANTI-IDIOTYPES, AND MOLECULAR MIMICRY
[9]
meric IgM alone and in complex with IgG anti-Id, respectively (K. H. Roux, T. J. Elliott, and M. J. Glennie, unpublished data). In some instances, IgM monomers may be more appropriate targets for complexing with anti-Id antibody. To avoid misinterpretation, care must be taken to ensure that the IgM monomers derived from reduced and alkylated pentamers retain intact heavy-heavy and heavy-light interchain disulfide bonds (Fig. 9C). 7 As is dramatically shown in Fig. 9D, the noncovalent forces holding a partially reduced IgM monomeric subunit together in solution are insufficient to maintain proper configuration or in some instances to keep the two heavy-light halves of the molecules together following interaction with an IgG anti-Id antibody. Acknowledgments I would like to thank Drs. N. Greenspan, J. Davie, T. Elliott, and M. Glennie for use of unpublished material and T. Fellers for reviewing the manuscript. This work was supported by National Institutes of Health Research Grant AI16596.
[9] U s e o f I m m u n o b l o t t i n g to D e t e c t I d i o t y p i c D e t e r m i n a n t s on Monoclonal Antibodies B y C . P E T I T , M . E . SAURON, M . GILBERT, a n d J . T H ~ Z E
Introduction A technique for the transfer of proteins from gels to paper has been developed ("protein blotting"). 1,2Three steps are involved: (1) separation of proteins by gel electrophoresis in the presence of urea, ampholytes, or sodium dodecyl sulfate (SDS), (2) transfer of these proteins to diazobenzyloxymethylcellulose (DBM) paper I or to nitrocellulose filters by electrophoresis or diffusion, 2,3 and (3) detection of the adsorbed proteins by incubation of the filter with specific reagents. The proteins can be i H. A. Erlich, J. R. Levinson, S. N. Cohen, and H. D. Devitt, J. Biol. Chem. 254, 12240 (1979). 2 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). 3 B. Bowen, J. Steinberg, U. K. Laemmli, and H. Weintraub, Nucleic Acids Res. 1, 1
(1980). METHODS IN ENZYMOLOGY,VOL. 178
Copyright© 1989by AcademicPress, Inc. All rightsof rcproductinnin any form reserved.
[9]
IMMUNOBLOTTING OF IDIOTYPES ON MONOCLONAL A b
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identified by the binding of specific antibodies 2 or by their ability to specifically bind ligands such as DNA, RNA, or histones. 3 This latter property indicates that proteins first separated on SDS gel and then transferred to nitrocellulose filters by diffusion have been at least partly renatured. In this chapter, we report on the utilization of the protein-blotting technique to study the idiotypic determinants expressed by an antipoly(Glu6°-Ala3°-Tyr1°) (GAT) monoclonal antibody (G5). Using polyclonal anti-idiotypic sera, two idiotypic specificities in particular have been studied on anti-GAT hybridoma products (HP): (I) the public idiotypic specificity (p. GAT) present in an identical form in all anti-GAT HP and all anti-GAT antibodies of every mouse strain tested 4 and (2) the highly conserved idiotypic specificity (h.c. GAT) also present on rat and guinea pig anti-GAT antibodies. 5 Thesetwo idiotypic specificities can be detected on blotted G5 molecules, previously run on a SDS-polyacrylamide gel. For this study, protein transfer was performed essentially according to the technique described by Bowen et al. 3 However, to favor the renaturation of idiotypic determinants of the blotted proteins and to minimize nonspecific binding, we have introduced some modifications of the method.
Materials
Monoclonal lmmunoglobulin, Antisera, and Purified Antibodies
Hybridoma cell line G5Bb2-2 was obtained by fusion of GAT-sensitized BALB/c spleen cells with myeloma cell line X-63 Ag8-NS-1. The corresponding HP (G5) (yl, r) was purified on GAT-Sepharose beads. 6 MOPC-21 (yl, K) protein was given by Dr. Juy. Rabbit anti-mouse Ig immune serum (R anti-M Ig) was given by T. Ternynck. Purified sheep anti-rabbit Ig antibodies free of cross-reactivity with mouse Ig were prepared as previously described 7 and labeled with horseradish peroxidase (PO) using a two-step procedure. 8 Normal BALB/c immunoglobulins were purified from normal BALB/c ascitic fluid as previously described. 9 4 G. Somm6, L. Leclercq, C. Petit, and J. Th6ze, Eur. J. Immunol. 11, 493 (1981). 5 G. Somm6 and J. Th6ze, Mol. lmmunol. 16, 1119 (1979). 6 L. Leclercq, J. C. Mazi6, G. Somm6, and J. Th6ze, Mol. lmmunol. 19, 1001 (1982). 7 C. Petit, J. C. Antoine, and S. Avram6as, lmmunochemistry 14, 479 (1977). s S. Avram6as and T. Ternynck, Immunochemistry 9, 1175 (1971). 9 j. Th~ze and G. Somm6, Fur. J. lmmunol. 9, 294 (1979).
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[9]
Anti-Idiotypic Sera The anti-idiotypic immune serum 715-7A4 was raised in a rabbit against BALB/c anti-GAT antibodies. The specificity and the characterization of this serum have been reported. 9 The serum 715-7A4 recognizes the p. GAT idiotypic specificity on the monoclonal BALB/c anti-GAT antibodies. 6 The anti-idiotypic immune serum 13-7A1 was raised in a rabbit against F.344 rat anti-GAT antibodies. The properties of this serum have been analyzed; the serum recognizes the h.c. GAT idiotypic specificity on monoclonal BALB/c anti-GAT antibodies. ~° Procedure
Gel Electrophoresis and Protein Transfer SDS-polyacrylamide gel electrophoresis was carried out as described by Laemmli. n Samples containing 10/~g of protein were diluted in "sample buffer" with or without 2-mercaptoethanol. Nonreduced samples were allowed to migrate on gels containing 7.5% acrylamide and 0.4% methylbisacrylamide. Reduced samples were separated on gels containing 15% acrylamide and 0.4% methylbisacrylamide. Immediately after electrophoresis, the SDS gel was shaken in transfer buffer (50 mM NaC1, 2 mM Tris-HCl, pH 7.4, 0.I mM phenylmethylsulfonyl fluoride) for 15 min at 20 °. The gel was then sandwiched between two sheets of nitrocellulose filters equilibrated in transfer buffer and two Whatman 3 MM filter papers and pressed together in a holder as described by Bowen et al. 3 The holder was immersed for 18 hr in 2 liter of transfer buffer under agitation at room temperature. After transfer, the nitrocellulose sheets were incubated for 30 min at 20°, with shaking, in phosphate-buffered saline (PBS: 10-z M phosphate buffer, pH 7.4, 0.15 M NaCI) containing 3% Nonidet P-40 (NP-40), then rinsed (3 times, 5 min each) in PBS. The nitrocellulose filters were finally incubated for 1 hr at 37° in saline buffer (0.9% NaCI, 10 mM Tris-HC1, pH 7.4) with 3% bovine serum albumin (BSA) to saturate the remaining protein binding sites of the nitrocellulose filters. This was followed by a brief washing in saline buffer containing 1%o Tween. The protein transfer was checked by staining of a strip of the nitrocellulose filters with Amido Schwarz 12(amido black) and staining of SDS gels with Coomassie blue. to C. Petit, M. Gilbert, G. Somm6, J. C. Mazi6, M. Dorf, and J. Th~ze, Mol. Irnmunol. 19, 1139 (1982). n U. K. Laemmli, Nature (London) 2,27, 680 (1970). 12 W. Schaffner and C. Weissmann, Anal. Biochem. 56, 502 (1973).
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Binding of Immune Reagents and Detection of Anti-PO Activity Strips cut from the nitrocellulose filters were incubated for 45 min in the presence of either rabbit anti-idiotypic serum, or R anti-M Ig serum, or normal rabbit serum (NRS). The anti-idiotypic sera were first diluted in NRS (v/v) and further diluted in PBS containing 1% Tween and 3% BSA. The R anti-IgM serum and the NRS were diluted in PBS, I% Tween, 3% BSA. The R anti-lgM serum was used at 1/200 dilution, the NRS at 1/20 dilution. All incubations were performed in small, heat-sealed plastic bags, so as to minimize the volume of reagents. Thereafter, the strips were rinsed (3 times, 10 min each in washing buffer (0.5 M NaCI, 10 mM Tris-HCl, pH 7.4, 1%o Tween) at 20° with shaking. PO-labeled sheep antirabbit Ig antibodies were then allowed to react with rabbit Ig, in PBS, 1% Tween, 3% BSA. After this last incubation, the strips of nitrocellulose were rinsed in the washing buffer. Strips were incubated for 5 min in the PO assay buffer (0.1 mM TrisHCI, pH 7.6), then placed in the same buffer containing 500/zg/ml of 3,3'diaminobenzidine (DAB) (Prolabo, Paris) and 1/zl/ml of H202 (30%). The enzymatic reaction was stopped at various times ranging from a few seconds to 5 min by immersion of the strips in water containing 0. I N HCI.
Detection of Conformational Idiotypic Specificities on a BALB/c Anti-GAT Antibody (GS) After migration on a SDS-polyacrylamide gel under nonreducing conditions and transfer by diffusion to a nitrocellulose filter, the monoclonal BALB/c anti-GAT antibody G5 was stained by the anti-idiotypic serum 715-7A4 (Fig. 1). Within 20 to 30 sec, an intense precipitate of DAB was formed when the anti-idiotypic serum was used at 1/20 dilution and the sheep anti-rabbit immunoglobulin antibodies at 40/~g/ml. Under the same experimental conditions, normal BALB/c immunoglobulins were only very weakly stained. Similarly, the MOPC-21 myeloma protein, which possesses a yl heavy chain and a r light chain like G5, was very faintly stained after incubation with serum 715-7A4. The ability of the anti-idiotypic serum 13-7A1 to specifically bind the G5 molecules after transfer was also tested. As shown in Fig. 1, after incubation with serum 13-7A1, intense staining of the G5 molecules was observed while very faint staining of normal BALB/c or MOPC-21 immunoglobulins was noted. This staining appeared in a few seconds when a 1/20 dilution of serum 13-7A1 was used with a concentration of anti-rabbit immunoglobulin antibodies of 40/xg/ml. The specificity of this idiotypic binding was checked. When G5 was incubated in presence of NRS, no staining of G5 by the sheep anti-rabbit immunoglobulins antibodies was observed (Fig. 1).
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[9]
FIG. 1. Detection of p. GAT and h.c. GAT idiotypic specificities on a monoclonal BALB/c anti-GAT antibody, G5. G5 (3'~, K), MOPC-21, and purified normal BALB/c immunoglobulin (NIg) were allowed to migrate on an SDS-polyacrylamide gel under nonreducing conditions. They were then blotted onto a nitrocellulose filter and incubated with 715-7A4, 13-7A1, or NRS. The enzymatic staining was stopped after 30 sec. T w o additional bands with apparent molecular weights slightly lower than G5 w e r e heavily stained b y s e r u m 715-7A4 and serum 13-7A1. These r e p r e s e n t e d v e r y minor c o m p o n e n t s as judged by Amido Schwarz (amido black) staining of the strips or C o o m a s s i e blue staining of the gels before blotting. T h e y m a y represent the products of a limited proteolysis or a n o n h o m o g e n e o u s denaturation by SDS, or heterogeneous glycosylation 13 of protein G5. The v e r y w e a k staining o f normal B A L B / c and MOPC-21 immunoglobulins b y serum 13-7A1 and 715-7A4 m a y be due to v e r y small amounts o f anti-isotypic antibodies in these sera. Optimal detection of the serum 715-7A4 and serum 13-7A1 bindings was obtained with the serum and antibody concentrations reported above. H o w e v e r , lowering the concentration o f b o t h reagents (up to 1/100 dilution for the immune serum and 10/zg/ml for the labeled antibodies) only slightly decreased the detection of the idiotypic determinants. 13R. Pitt-Rivers and F. S. A. Umpiom-Bato, Biochem. J. 109, 825 (1968).
[9]
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Comments
Some technical points have to be discussed. First, transfer by electrophoresis (for 1.5 hr, under 200 mA, in 192 mM glycine, 25 mM Tris buffer) was less efficient than transfer by diffusion. This was judged by Amido Schwartz staining of blotted G5 molecules and may be explained by the fact that nitrocellulose paper binds molecules moving under an electric field less efficiently. Second, an increase of the binding of the two antiidiotypic sera to the blotted G5 molecules was obtained by incubation of blotted proteins in NP-40 immediately after the transfer; this result can be attributed to the effect of NP-40 on the renaturation of proteins previously denatured by SDS as reported by Ames and Nikaido.14 Finally, the significant nonspecific binding to the nitrocellulose filters observed in preliminary experiments was prevented by addition of 1% Tween in the incubation and washing buffers. Similarly, washing with 0.5 M NaCI eliminates the nonspecific binding. As a general rule, both specific heavy and light chains are required for the expression of idiotypic determinants. However, in some cases, idiotypes have been detected on isolated chains. 15 We have attempted to detect the p. GAT and the h.c. GAT idiotypic specificities on H5 and L5 separated chains after protein transfer. We failed to reveal the p. GAT or the h.c. GAT specificities on H5 or L5 using the same experimental conditions permitting their detection on whole G5 molecules. Preincubation of the separated chains with NP-40 did not change the results. Thus, the association of H5 and L5 chains is necessary for the expression of the p. GAT and the h.c. GAT specificities. These results have been confirmed by Somm6 et al.16 They observed that isolated H5 and L5 chains do not inhibit the binding of serum 715-7A4 and serum 13-7A1 to radiolabeled G5 molecules. Thus, the results obtained by the protein-blotting technique and radioimmunoassay are in complete agreement, and demonstrate that p. GAT and h.c. GAT are conformational idiotypic specificities. The ability to detect conformational idiotypic determinants on molecules previously migrated on SDS gels, under nonreducing conditions, indicates that protein blotting followed by the treatment described above allows a satisfactory renaturation of blotted proteins.
14j. F. L. Ames and K. Nikaido, Biochemistry 15, 616 (1976). l~ A. Nisonoff, J. E. Hopper, and S. B. Spring, "The Antibody Molecule," p. 481. Academic Press, New York, 1975. 16 G. Somm6, J. Rocca-Serra, L. Leclercq, J. C. Mazi6, L L. Moreau, D. Moinier, M. Fougereau, and J. Th~ze, Mol. Immunol. 19, 1010 (1982).