Antibodies of restricted heterogeneity induced by DNP-insulin

Immunoc/tem/atry, 1973, Vol. 10,pp. 331-335.

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ANTIBODIES OF RESTRICTED H E T E R O G E N E I T Y I N D U C E D BY D N P - I N S U L I N * t K L A U S KECK~, A L L A N L. G R O S S B E R G and D A V I D P R E S S M A N Department of Biochemistry Research, Roswell Park Memorial Institute,§ Buffalo, New York 14203, U.S.A. (First received 27 August 1972; in revised form 30 October ! 972)

Al~ract-lmmunization of rabbits with a preparation of DNP-insulin, specially purified by isoelectric focusing, has !ed to the production of anti-DN P antibody of very restricted heterogeneity in all seven rabbits examined and in amounts up to 800/zg/ml serum. The homogeneity of one of the antibody samples was shown by the straight line character of its binding curve for =H-labeled DNPlysine (Sips' heterogeneity index = 1-0) and by the simplicity of its isoelectric focusing pattern. Similar simple patterns were observed for antibodies from the other six rabbits although two produced very little anti-DNP antibody. INTRODUCTION When a rabbit is injected with a hapten-protein conjugate, the anti-hapten response is often one of rather limited heterogeneity, i.e. a relatively small number of different antibodies are produced, rather than a very heterogeneous response in which a large number of antibodies are produced, with binding constants distributed in Gaussian fashion around a common mean (Pressman et al., 1970). Even in the case of rabbits injected with very complex antigens such as diazotized p-aminobenzoic acid or diazotized p-aminobenzenearsbnic acid coupled to bovine gamma globulins, where the protein carders are a mixture of antigens and the haptenic groups are present in different environments on different antigen molecules due to differences in positions of coupling, there is often still a response of very restricted heterogeneity. Indeed, sometimes the response is to produce essentially homogeneous anti-hapten antibody (Roholt et al., 1970; Seon et al., 1972). It would appear from this that although the rabbit may potentially be able to make a large number of different antibodies against the same common antigenic group, only a few of the cells which could produce antibodies are stimulated for antibody

*This paper was supported in part by Grant No. A1-10454 from the National Institute of Allergy and Infectious Diseases and Grant No. CA-11656 from the National Cancer Institute. tAbbreviations: BBS: borate buffered saline, pH 8; DNP: 2,4-dinitrophenyl. $Visiting Research Associate. On leave of absence from the University of Konstanz, Konstanz, Germany and supported by the Deutsche Forschungsgemeinschaft. Present address: University of Konstanz, Department of Immunology, Konstanz, Germany. §A unit of the New York State Department of Health.

production and of these, certain ones quickly predominate so that the antibody produced appears to be relatively homogeneous (Pressman et al., 1970). it also appears that use of a simple antigen in which there are only one or two hapten groups in a constant environment should greatly influence the degree of homogeneity of the antibody response. On this basis, Eisen er al. (1964) studied the response to D N P coupled to ribonuclease, Little and Counts (1969), the response to D N P coupled to insulin, Brenneman and Singer (1968) carried out studies with D N P coupled to the single SH group of papain, and Montgomery et al. (1972) studied di-DNP-gramacidin-S. Only the last authors reported obtaining antibodies with limited heterogeneity in appreciable amounts. We have carried out further experiments with D N P coupled to insulin in spite of the report of Little and Counts (1969) that the antibodies they obtained were heterogeneous. We obtained responses of extremely limited heterogeneity in rabbits receiving specially purified D N P - i n s u l i n . It may well be that the heterogeneity report by Little and Counts (1969) was due to the presence of several DN P impurities in the insulin. In the experiments reported here the D N P insulin was purified by isoelectric focusing and the antibody response that we observed using the purified material as an antigen indicated very limited heterogeneity. MATERIALS AND METHODS

Insulin, Zn free, was a gift of Dr. Y. Yagi of this laboratory. It was prepared by repeated isoelectric precipitation of insulin at pH 5.5 in the presence of ethylenediamine. Glass plates for electrofocusing were from IIford Ltd., llford, Essex, England. Acrylamide, NNmethylenebisa~crylamide and riboflavin were purchased from Bio-Rad Laboratories, Richmond, Va. Ampholine carders were from LKB Products, Rockville, Md. 331

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KLAUS KECK, A L L A N L. G R O S S B E R G and D A V I D PRESSMAN

Ultra pure urea was from Schwarz-Mann, Orangeburg, N.Y. and the glutaraldehyde (biological grade) was from Fischer Scientific Co., Pittsburg, Pa.

Preparation of D N P-insulin Insulin, Zn free, in 0.035 M Tris-phosphate bufered 6 M urea, pH 9, was first purified by chromatography on DEAE cellulose equilibrated with the same buffer. Elution was begun with this buffer; later the pH of the buffer was lowered to 8-5 and then to 7.9. The insulin came off with the last buffer. The main fraction was dialyzed against distilled water for several days during which process it precipitated, it was finally lyophylized. Dinitrophenylation was carried out as described by Li (1956). To 200rag of purified insulin in 35ml 0 . 1 M sodium carbonate was added 0.5 g of DNP-sulfonic acid and the pH was adjusted to 11 with 1 N sodium hydroxide. The solution was left in the cold room for nine days. The crude DNP-insulin was separated from the reaction mixture by precipitation at pH 5.5 and centrifugation. This procedure was repeated two or three times until the supernate remained colorless. After dialysis against several changes of distilled water, the product was dissolved in BBS at pH 8.6. Preparative isoelectric purification of D N P-insulin The electrofocusing mixture was made up as described elsewhere (Keck et al., 1972), so that the final concentration of acrylamide was 5%. Ampholine pH 3-6 was 2% and urea was 6 M. To 50 ml of this solution, 2 ml of the crude D N P insulin solution containing about 10-20 mg DNP-insulin was added and the gel was photopolymerized for I hr between a chromogelatin coated glass plate and a plexiglass plate with two plexiglass spacers keeping the two plates about 1.5 mm apart. The glass plate and attached gel was put in a humidified chamber in the cold room on carbon rods as described by Awdeh et ai. (1968). The gel was first run at a constant current of 2 mA. After reaching 400--500 V it was run at a constant voltage (400V) for 12-24hr. The plate showed about 5-6 light-scattering bands, some of which were yellow. The main band was very sharp, intensely yellow, and about 2 mm wide. This band was cut out with a scalpel and eluted 2-3 times with BBS. The eluate was dialyzed against BBS. The protein concentration was estimated by the Folin method, and its purity checked by polyacrylamide gel electrofocusing according to the procedure described by Hoffman et al. ( 1971 ). Injection of rabbits Three rabbits, No. 5779, 5780 and 5781 were each injected with 1 mg of the purified DNP-insulin in complete Freund's adjuvant in the footpads and intraderreally on Day I, Day 21 and Day 30. Sera were collected twice a week. A second group of four rabbits was injected and bled similarly but at a later time. Electrofocusing of anti-DNP antisera in thin acrylamide gel plates The general method has been described in detail by us (Keck et al., 1972). Usually a 5% polyacrylamide gel was used containing 3 M urea and 2% ampholine pH 3-10 or pH 5-8. The sera were applied directly to the plate on a small strip of filter paper. After electrofocusing, the gamma globulins were immobilized in the following way: The plates were treated with 18% sodium sulfate solution

for 2 hr, then 1 ~,1 of 50% glutaraldehyde was added per ml of sodium sulfate solution and allowed to react for ! hr. After rinsing with tap water, the plates were washed in BBS containing I mg/ml sodium hydride for 5-10 hr, to reduce unreacted groups (Keck et al., 1972).

Radioiodination of D NP-ovalbumin The procedure described by Yagi (1971) was used. Usually 100~tg of the protein was labeled with !-2 mCi of nSl. Labeling of the separated bands with J2Sl-labeled D N P ovalbumin A plexiglass plate was placed on a level surface. The electrofocused plate was supported over it on two plexiglass spacers I mm thick with the polyacrylamide surface facing down. The space between the plates was filled with a solution of the nSI-labeled DNP-ovalbumin, e.g. 50 p,Ci (usually 1 mCi/100 mg DNP-ovalbumin) diluted to 30 ml with 0.5% ovalbumin in BBS. This solution had been evacuated for a few minutes to prevent formation of air bubbles. The plates were covered with a plastic box to prevent evaporation. After incubation overnight, the plates were removed, rinsed with tap water and soaked for 24 hr in saline containing 10% BBS. After rinsing again with tap water, the plates were dried at room temperature overnight. After washing there was essentially no visualized background radioactivity on the plate. The dried plates were exposed to Kodak No-Screen Medical Film in an X-ray exposure holder and after 1-3 days developed with X-ray developer. Contact prints were made from the radioautograph. Equilibrium dialysis Binding constants of anti-DNP antibodies were determined by equilibrium dialysis (Pressman and Grossberg, 1968) using SH-labeled ¢-N-DN P-lysine as ligand. Antibody content of serum The antibody concentration of the serum of rabbit 5779 was determined by equilibrium dialysis and found to be 800/~g antibody protein per ml of original serum. The other sera appeared to be of comparable or lower antibody concentration as estimated from the intensity of the isoelectric focused bands. ~TS T h e D N P - i n s u l i n we p r e p a r e d b y t h e m e t h o d o f Li (1956) was h e t e r o g e n e o u s w h e n e x a m i n e d b y isoelectric f o c u s i n g in a small gel c o l u m n . O v e r five p r o t e i n b a n d s were s e e n (Fig. I A). When the fraction obtained by cutting out the main p r o t e i n b a n d o f t h e large scale p r e p a r a t i o n was a s s a y e d b y e l e c t r o f o c u s i n g it s h o w e d a single p r o t e i n b a n d (Fig. I B). T h e single b a n d c h a r a c t e r o f this r e f o c u s e d p r o t e i n d e m o n s t r a t e s the purity o f t h e p r o d u c t a n d also i n d i c a t e s t h a t the o t h e r protein b a n d s in Fig. I A are not c a u s e d b y p h o t o lytic a l t e r a t i o n o f t h e protein d u r i n g p h o t o p o l y m e r i z a t i o n o f t h e gel, as might h a v e o c c u r r e d b e c a u s e o f the p h o t o s e n s i t i v i t y o f D N P a m i n o acids. T h i s f r a c t i o n o f D N P - i n s u l i n was i n j e c t e d into r a b b i t s with c o m p l e t e F r e u n d ' s a d j u v a n t o n d a y !, 21 a n d 47. T h e c o l l e c t e d sera w e r e e l e c t r o f o c u s e d

Antibodies of Restricted Heterogeneity induced by DN P-insulin

8

15

22

27

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3G

42

45

Days after first injection Fig. 2. Anti-DNP response of rabbit No. 5779 at the indicated days after initial immuniTation with purified DNP-insulin. The electrofocusingpatterns were obtained with whole serum. The antibodies were visualized by the specific fixation of nSl-DNP--ovalbumin followed by radioautography. Note continued production of the three main components.

A

B

Fig. 1. Column gel electrofocusing of DNP-insulin. A: Initial product from coupling reaction. B: Purified product used for immunization. in polyacrylamide gel, the gamma globulins immobilized with glutaraldehyde after precipitation with sodium sulfate, as described. Anti-DNP antibodies were specifically visualized by treating the gel with iUl-labeled DNP-ovalbumin. Contact prints of the autoradiographs are shown in Fig. 2. Figure 2 shows the development of the antiDNP response of rabbit No. 5779, the first which responded to the immunization. The first bleeding showing anti-DNP antibodies was obtained on day 22. It showed three major bands and some minor bands. As the figure shows, these bands intensified during the following weeks. The response resembles those reported by Montgomery et al. (1972) with di-DN P-gramaciden-S. Although it has not been proven that the three bands reflect only one clone, the banding patterns look very similar to those obtained with myeloma proteins which usually consist of two to five main bands. To investigate the heterogeneity of the antibody in the serum from Day 42 (antibody content was about 800 ~g/ml) the gamma globulin fraction was precipitated with sodium sulfate and was sub-

mitred to equilibrium dialysis against tritiated DNP-lysine. The Sips' plot of the binding is shown in Fig. 3. The heterogeneity index of the antibodies is about one, indicating that the different bands either reflect one clone or different clones with the same or very close to the same binding constant and that the additional weaker bands probably do not contribute much to the binding. Figure 4 shows typical isoelectric focusing patterns which demonstrate the limited heterogeneity of the antibodies produced by five out of seven of the rabbits tested. The sera of the two remaining rabbits showed only very weak bands even 40 days after the primary injection. None of the sera showed the usually observed heterogeneous antibody response. The restricted heterogeneity shown by the isoelectric focusing patterns is not an artifact produced by the method of immobilizing the antibodies in the gel with glutaraldehyde, and binding antigen to the antibody. We have previously shown (Keck et al., 1972) that antibody globulin in acrylamide gel, treated with glutaraldehyde by our procedure and then assayed in the gel for antibody binding activity by equilibrium dialysis, gives results for binding constant and number of binding sites present, which are in good agreement with such values obtained for a portion of the same antibody globulin preparation in free solution, not treated with glutaraldehyde and assayed for binding parameters by the conventional method of equilibrium dialysis.

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KLAUS KECK, ALLAN L. GROSSBERG and DAVID PRESSMAN IO

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Fig. 3. Sips' plot for binding of "~H DNP-lysine to antibody in the y-globulin fraction of rabbit No. 5779. The slope of about one indicates essential homogeneity. ,40 (total antibody site concentration) was taken as 9.5 x 10-e M as determined from a Scatchard plot of b/c vs b (b = molar concentration of bound hapten; c = molar concentration of free hapten). The calculated value of the binding constant, K 0 - - 7 x 10eM-1. The antibody site concentration found corresponds to antibody protein concentration of 800/~g/ml of original serum.

5779 DISCUSSION Little and Counts (1969) injected guinea pigs with D N P - i n s u l i n prepared according to Li (1956). Using enzyme digestion and amino acid analysis they showed that only lysine 29, the single lysine residue of the molecule, was labeled with the D N P group. After injecting guinea pigs with this product they reported that they found no restriction in the heterogeneity of the antibody response as compared to the response to D N P - b o v i n e gamma globulin. The heterogeneity index of the anti-DNP antibodies was found to be 0.59. As shown in the present study, the D N P - i n s u l i n we prepared according to the procedure described by Li (1956) consisted of several components which were resolved by electrofocusing even though the insulin we used had been purified so that it showed only one band in electrofocusing. The D N P - i n s u l i n we used for preparing antibodies was the main protein band obtained following preparative electrofocusing. The product gave a single band on refocusing. No further analysis was carried out with this material. We assume according to the data of Little and Counts (1969) that this is mono-e-N-lysine 2 9 D N P - i n s u l i n . This material was injected into rabbits and it induced antibodies with highly restricted heterogeneity in concentrations up to 800/.tg antibody/ml (Fig. 3). The patterns observed (Fig. 4) indicate to us that well over 95 per cent of the antibody present was of a highly restricted nature since only one or two components are evident. We estimate that indivi-

5 7 8 0 5781

Rabbit

5804 5807

No.

Fig. 4. Limited heterogeneity of anti-DNP antibodies produced by different rabbits. Electrofocusing patterns were obtained as described in Fig. 1. Note the apparent limited heterogeneity patterns. Note also that the patterns for different rabbits were not made simultaneously on the same plate and therefore no significance can be placed on the relative positions of the bands. dual additional components would have to be present in at least I per cent of the total to be seen. It would appear that a purified antigen such as we used provides a means for obtaining enough homogeneous antibody to permit detailed structural studies. The heterogeneous response observed by Little and Counts (1969) was probably due to the heterogeneity of the D N P - i n s u l i n they used in view of the fact that we obtained very restricted responses with the purified antigen. We did not test whether the unpurified D N P - i n s u l i n gave a heterogeneous response in our hands.

Acknowledgements-We wish to thank Messrs. Leonard Rendina, Jake Planinsek and Arthur J. Trott for their help with the experiments. REFERENCES

Awdeh Z. L.. Williamson A. R. and Askonas B. A. (1968) Nature, Lond. 219, 66.

Antibodies of Restricted Heterogeneity induced by DNP-insulin Brenneman L. and Singer S. J. (1968) Proc. natn. Acad. Sci. U.S.A. 60, 258. Eisen H. N., Simms E. S., Little J. R. Jr. and Steiner L. A. (1964) Fedn Proc. 2,3, 559. Hoffman D., Grossberg A. L. and Pressman D. (1971) J. lmmun. 107, 325. Keck K., Grossberg A. L. and Pressman D. (1972) Ear. J. lmmun. (in press). Li C. H. (1956) Nature, Lond. 178, 1402. Little J. R. Jr. and Counts R. B. (1969) Biochemistry 8, 2729. Montgomery P. C., Rockey J. H. and Williamson A. R.

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(1972) Proc. natn. Acad. Sci. U.S.A. 69, 228. Pressman, D., Grossberg, A. L. (1968) Structural Basis of Antibody SpecO~city, Benjafffm, New York, N.Y. Pressman D., Roholt O. A. and Grossberg. A. L. (1970) Ann. N.Y. Acad. Sci. 169, Art. !, 65. Roholt O. A., Seon B.-K. and Pressman D. (1970) lmmunochemistry 7,329. Seon B.-K., Roholt O. A. and Pressman D. (1972) J. lmmun. 108, 86. Yagi Y. (1971) Methods in Immunology and lmmunochemistry (Edited by Williams and Chase), Vol. 3, p. 463-475. Academic Press, New York.