Antibodies to relatively homogeneous haptens: The temporal pattern of their antigen-binding energies

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BBA 3 I 009

Antibodies to relatively homogeneous haptens: the temporal pattern of their antigen-binding energies Antibodies to randomly substituted hapten-protein complexes show change in their average binding energies as a function of time. Shortly after immunization, they m a y have a relatively low average affinity for the antigen. Over a period of weeks the average affinity rises 1-4, and increases of up to four orders of magnitude have been recorded 1. 7A antibody of horse is reported to have a higher association constant (KA) than 7G early in the course of immunization but variations with time could not be examined 2. Such changes in the distribution of antibody population with time are an aspect of the structural heterogeneity which is a characteristic of most antibodies so far studied s. Current studies by the authors are aimed at examining to what extent the structural heterogeneity of anti-hapten antibodies is a function of the heterogeneous environment of the haptenic group of the immunogen. The antibody-combining site is substantially larger than most of the small aromatic determinants used as haptens 6. Consequently, the site must include not only the hapten but a variety of combinations of hapten and surrounding structures. Also, haptens in juxtaposition on the protein surface m a y give rise to different antibodies than single haptense, 7. In order to overcome some of the antigenic heterogeneity inherent in randomly substituted protein-hapten complexes, a defined sequence macromolecular peptide of general formula has been synthesized: re-DNP-L-Lys-(D-Ala-L-Ala)571o. 2 (average)

This peptide has an average mol. wt. of about 1OLOO. In this compound the D N P hapten is in a relatively homogeneous environment, the haptenic groups are separated b y a distance of about 30 A when the peptide is the extended form, a distance larger than present estimates of the size of the antibody combining site. Moreover, the peptide is a random coil in solution v. The average association constants of the antibodies to this material were examined by equilibrium dialysis at various times after the initial immunization and in some animals after secondary boosting. Eleven New Zealand white rabbits were immunized b y subcutaneous injection of the antigen in complete Freund's adjuvant into the toe pads. All animals received an initial dose of 1.o mg except rabbit 3 who received 0.5 mg. All rabbits except 3 received a 0.5 mg boost of the polymer in 0. 9 % saline intravenously at 15 weeks, and rabbits 6- 9 received a third boost of 0.5 mg polymer intravenously at 20 weeks. Eight out of nine animals produced antibodies reacting with DNP-L-lysine in quantities from 3O-lOOO/zg antibody protein per ml serum. [3H]DNP-L-Lysine was prepared from [3HlFDNB (Nuclear Chicago) 8. The final specific radioactivity of the chromatographically purified product varied in different preparations from 7.4-9.2 C/mmole. With these specific radioactivities it is possible to assay accurately association constants up to lO 1° M-1. The 7G fraction was isolated from individual bleedings by the method of CAMPBELL 9, and contained 8o-9o % of the antibody in whole serum. Equilibrium dialysis was carried out at 2o ° using the method described by EISEN 1°. In each experiBiochim. Biophys. Acta, i4o (i967) 558-560

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ment a control was run to estimate non-specific binding employing similar concentrations of 7G from non-immunized animals. Non-specific binding of the hapten was l o w - - b e t w e e n 2-5 % of the total counts bound b y the antibody. The results, which are shown in Fig. i, indicate that the association constants of the first detectable antibodies are high. At 2. 5 and 3 weeks they are of the order of 1.3-4.5.1o 9 M-1 although one was as high as 33.1o 9 M-1. There was no rise in association constant with time. A typical response was that of the rabbit 2 which showed a K A of 2.I" 109 M-1 at 21 days, 3.4" lO9 M-1 at 49 days, I . I . lO9 M-~ at lO5 days, and 1.6. lO9 M-~ at 171 days. Equilibrium dialyses were carried out at higher hapten concentrations in order to detect low-affinity antibody, but none was found.

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l~ig. I. P l o t of log K a vs. t i m e in d a y s following i m m u n i z a t i o n . T h e n u m b e r s refer to i n d i v i d u a l r a b b i t s i m m u n i z e d w i t h t h e defined s e q u e n c e p o l y m e r . B e s t l e a s t - s q u a r e s line t h r o u g h t h e s e points ( ) ; s t a n d a r d error (. . . . . ), A, B, C, ( . . . . . . ); 3 r a b b i t s i m m u n i z e d w i t h 5 m g of D N P - ( b o v i n e ?-globulin) f r o m EIsEN AND SISKIND 1.

The binding studies in these experiments have been carried out with the whole yG fraction, rather than with purified antibody. Purification of high affinity D N P antibodies generally results in a low yield and consequently the material available for study would not necessarily be representative of the range of antibodies produced. Previous studies using small doses of randomly substituted protein-hapten complexes have all demonstrated a rise of average association constants with time 1-4. The conditions under which our experiments were carried out are analogous to those of EISEN AND SISKIND1. These authors noted that small doses of immunizing agent --5.0 mg DNP-(bovine y-globulin)--gave the largest increase in association constant with time (see Fig. I), while large doses--25o mg DNP-(bovine y-globulin)--gave no such increase. However, in the experiments in which large immunizing doses of DNP(bovine v-globulin) were used, the antibodies produced were of low affinity throughout. This contrasts with the high-affinity antibodies produced against the defined sequence polymer in these experiments. The doses of immunizing polymer used here contained 30-60 % of the actual number of moles of hapten used by EISEN AND SISKIND 1, and resulted in approximately the same concentration of total antibody. I t seems likely, therefore, that the differences observed m a y be accounted for by differences in the antigens used and not b y different methods of immunization or different immunizing doses. From the data presented here, it is not possible to conclude that the observed constancy of K A with-time is in fact associated with a reduction of Biochim. Biophys. ,~cta, 14o (1967) 558-560

560

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structural heterogeneity in the antibody population. Investigations are in progress to clarify this question. Our thanks are due to Miss J A N E ROSNER and Mrs. FLORENCE GREEN for their enthusiastic and skillful technical assistance. This investigation was supported by Grants AI-o4967 and HE-o6664 of the U.S. Public Health Service; F . F . R . is an Established Investigator of the American Heart Association.

The Cardiac Unit, Medical Services, Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Boston, Mass. (U.S.A.)

F R A N K F . RICHARDS EDGAR HABER

H. N. EISEN AND G. W. SISKIND, Biochemistry, 3 (1964) 996. R. N. KLINMAN, J. H. ROCKEY, G. FRAUENBERGER AND F. KARUSH, J. Immunol., 96 (1966) 587 . L. A. STEINER AND H. N. EISEN, Bacteriol. Rev., 2 (1966) 383 . J- R. LITTLE AND H. N. EISEN, Biochemistry, 5 (1966) 3385 . J. B. FLEISCHMAN, Ann. Rev. Biochem., 35 (1966) 835. E. A. I~ABAT, J. Immunol., 97 (1966) i. F. F. RICHARDS, R. W. GLOANE JR. AND E. HABER, Biochemistry, 6 (1967) 476. 1R. R. PORTER AND F. SA~GER, Biochem. J., 42 (1948) 287. D. H. CAMPBELL, J. S. GARVEY, IN. E. CREMER AND H. ~). SOSSDORF, in D. H. CAMPBELL, Methods in Immunology, W. A. B e n j a m i n , Inc., N e w York, 1963, p. 122. io I-I. N. EISEN, in H. N. EISEN, Methods in Medical Research, Vol. IO, Y e a r b o o k Medical P u b lishers, Inc., Chicago, 1964, p. lO6. I 2 3 4 5 6 7 8 9

Received June 6th, 1967. Biochim. Biophys. Acta, 14o (1967) 558-56o