Immunologic Cross-Reaction Between Luteinizing Hormone and Human Chorionic Gonadotropin

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1 Immunologic Cross-Reaction Between Luteinizing Hormone and Human Chorionic Gonadotropin MELVIN L. TAYMOR, M.D., DONALD A. GOSS, M.D., and ALBERT BUYTENDORP, M.D. RECENTLY a number of reports 2 4 have indicated that it is possible to detect human luteinizing hormone ( LH) by means of a human chorionic gonadotropin ( HCG) antigen-antibody system. Pituitary and urine fractions with LH content inhibited the agglutination reaction between red cells or latex particles sensitized with HCG and HCG antiserum prepared in rabbits. An immunologic cross-reaction between HCG and LH has been theorized, but since commercial HCG preparations contain a large number of protein components, it is possible that this reaction may have been due to contamination of the HCG preparations with LH. It is the purpose of this report to present evidence that this reaction is indeed due to an immunologic cross-reaction. MATERIALS The antigens utilized in this study included ( 1) crude commercial HCG ( 2760 U.;mg.), ( 2) a relatively "pure" HCG assaying at approximately 8000 U. jmg., 0 and ( 3) human pituitary LH. Antisera were prepared in rabbits by the injection of these antigens combined with Freund's adjuvant. Latex particles of constant size were sensitized with HCG, both impure and "pure." From the Gyn-Endocrine Laboratory of the Peter Bent Brigham Hospital, Boston, Mass. This study was supported in part by Grants A-3759 and A from the National Institute of Health, U. S. Public Health Service, and in part by a grant-in-aid from the Ortho Research Foundation. The HCG preparations, the LH and HCG antisera, and the sensitized latex particles used in this study were generously supplied by Mr. William Pollock of the Ortho Research Foundation. The human pituitary LH was supplied by Dr. Ralph Reisfeld of Merck Sharp & Dohme. Immunoelectrophoretic studies described were carried out by Dr. Calvin Saravis of the Protein Foundation, Boston, Mass. Presented at the 19th Annual Meeting of the American Society for the Study of Sterility, New York, N. Y., Apr , *Product of Roussel Corporation, New York, N. Y. 603

2 604 TAYMOR ET AL. FERTILITY & STERILITY Biologic Inactivation Studies METHODS The effect of "pure" anti-hcg on the LH activity of human pituitary LH and human menopausal gonadotropin ( Pergonal-24) was studied by the ventral prostate assay. Samples of each preparation were incubated with an excess of anti-hcg for 1 hr. at 37 C., and the mixtures were then injected once daily for 4 days into hypophysectomized immature male rats. Mean ventral prostate weights were determined. Equivalent amounts of pituitary LH and Pergonal were similarly incubated with saline and likewise assayed. Six animals were utilized at each dose level. Latex Particle Agglutination Inhibition Studies Serial dilutions of LH were carried out in physiologic saline, with.5-ml. aliquots of these dilutions mixed with.5 ml. of HCG antisera and incubated for 1 hr. at 37 C. After 1 ml. of the suspension of latex particles sensitized with HCG was added, incubation was continued for 2 hr. at 37 C. Centrifugation at 2500 rpm ( 1g) for 2 min. was carried out, and the percentage of light transmission was measured in a Coleman photometer. Gel Diffusion Studies The gel diffusion studies were carried out according to a modification of the method described by Ouchterlony. A 4-mm. layer of 1% agar was poured into a petri dish and allowed to solidify. Wells were cut.5 em. square and.5 em. apart, forming an equilateral triangle. Antiserum was placed at the apex of the triangle and the two antigens to be studied placed in the 2 wells at the base of the triangle. The plates were kept in a humid atmosphere at 37 C. and read for precipitin lines at the end of 72 hr. Immunoelectrophoresis Studies Immunoelectrophoresis was carried out on glass slides also covered with a thin film of 1% agar, and in which suitable wells and troughs had been cut with a plastic template. After the antigen had been placed in the center well, immunoelectrophoresis was carried out in a barbital buffer at 0.1 ionic strength and at a ph of 8.2 according to the method described by Crowle. The current used was 8 v ;em. for 2 hr. The antisera were then added, and immunodiffusion was allowed to occur at 5 C. for hr. When satisfac-

3 VoL. 14, No. 6, 1963 LH AND HCG CROSS-REACTION 605 tory precipitin lines had appeared, the slides were washed for 2 days in physiologic saline, immersed in alkaline distilled water for one day, stained with a triple dye, washed with 2% acetic acid, mounted, and read. RESULTS Figure 1 demonstrates a comparison between the immunoelectrophoretic patterns of the commercial preparation of HCG ( HCG-A-1) and that of the pure preparation (HCG-B-1). Antiserum from the pure preparation had been placed in the outer and lower troughs (211-77), and antiserum from the crude preparation had been placed in the center trough ( ). It can be seen that the crude preparation contained at least 10 components. The pure HCG, on the other hand, seemed to be made up of primarily one component, although its homologous antiserum appeared to contain one and possibly two contaminants. These two preparations and their homologous antisera were used in the experiments. Biologic Inactivation The result of the biologic inactivation studies are shown in Table 1; 25 f.lg. of pituitary LH and 800 pg. of Pergonal-24 resulted in a five- to sixfold increase in ventral prostate weight. The biologic effect of these preparations was inactivated when the material was incubated with "pure" anti-hcg. Fig. 1. Immunoelectrophoretic patterns of crude HCG (A-1) and "pure" HCG (B-1) and their antisera.

4 606 FERTILITY TAYMOR ET AL. & STERILITY TABLE 1. Effect of "Pure" Anti-HCG on LH Effect of Human Pituitary LH and Human Menopausal Gonadotropin ( n=6) Material injected Controls 25 p.g. LH 800 p.g. Pergonal p.g. LH & anti-hcg 800 p.g. P-24 & anti-hcg Mean vent. prost. wt S.D Latex Particle Agglutination Inhibition Figure 2 demonstrates the effect of increasing concentrations of human pituitary LH on the turbidity of the latex particle HCG antigen-antibody system after critical centrifugation. Increasing concentrations of LH caused increasing inhibition of agglutination, and therefore, relatively more of the unagglutinated particles remain in suspension. Figure 3 demonstrates the increasing turbidity produced by the reaction of increasing concentrations of human pituitary LH in both a crude and a "pure" HCG system as measured in a Coleman photometer. Turbidity has been calculated as 100 minus the light transmission, and has been plotted against the concentration of LH on a log scale. Each curve is the mean of three independent dilutions of LH. It is apparent that LH reacted in a "pure" system as well as in an impure system. Gel Diffusion Figure 4 demonstrates the results of the precipitation reaction of "pure" HCG antiserum with "pure" HCG and with human pituitary LH. Good Fig. 2. Effect of increasing concentrations of human pituitary LH on turbidity of an anti-hcg-hcg latex particle system.

5 VoL. 14, No. 6, 1963 LH AND HCG CROSS-REACTION 607 precipitin lines formed between the antiserum and both antigens. An additional minor band precipitated between the HCG antigen and antiserum, indicating that this was only a relatively pure preparation. However, the 80 Fig. 3. Comparison of effect of human pituitary LH on turbidity of "pure" and impure HCG systems :~!------;J-!;-o----,2t<o----,4:';;-o----:'eo L.H. )"Vmo./ml. Fig. 4. Ouchterlony gel diffusion study of human pituitary LH and "pure" HCG against "pure" HCG antiserum.

6 608 TAYMOR ET AL. FERTILITY & STERILITY major band of precipitation joined with that from the LH to form the chevron of immunologic cross-reaction or possibly immunologic identity. Immunoelectrophoresis "Pure" HCG was studied in relation to its own antiserum and LH antiserum. Human pituitary LH was studied in relation to its own antiserum and "pure" HCG antiserum. The results are shown in Figure 5. In the studies with the "pure" HCG against its own antiserum one major precipitin line was produced along with a secondary line that once again indicated the presence of a minor contaminant. LH antiserum reacted against HCG to give a single band in the same position as the major one produced by the HCG antiserum. The lines appeared to fuse towards the anode pole. In the studies with LH antigen against its own antiserum, there was also evidence of a minor contaminant, but both HCG antiserum and LH antiserum produced bands in a similar relation to the LH antigen. However, the lines were not drawn out enough to demonstrate fusion. Fig. 5. Immunoelectrophoretic studies of "pure" HCG against anti-lh and anti-hcg, and of human pituitary LH against anti-lh and anti-hcg. DISCUSSION The fact that human pituitary LH caused agglutination in a "pure" HCG system as well as in an "impure" system suggests that the LH reacted immunologically with the HCG and not with LH that had contaminated the HCG preparation. This conclusion is dependent upon the acceptance of t~e relative purity of the Roussel HCG and its homologous antiserum. The im-

7 VoL. 14, No. 6, 1963 LH AND HCG CROSS-REACTION 609 munoelectrophoretic studies demonstrated a fairly marked degree of purity. There was indeed one minor contaminant, but the major precipitin band was the one that joined immunologically with the major precipitin band of the pituitary LH preparation. This was confirmed in the Ouchterlony gel study, where a minor contaminant is also noted, but once again the major precipitin line joined in the chevron of immunologic cross reaction with the LH. It would appear that by far the major hormone in the preparation is HCG itself, and therefore, the major component of the antiserum produced is anti HCG. Therefore, one can assume that it is anti-hcg that accounts for the biologic inactivation of the LH effect and not a contaminant. Verification of these conclusions awaits the development of even purer HCG and LH preparations and their homologous antisera. Peter Bent Brigham Hospital 721 Huntington Ave. Boston 15, Mass. REFERENCES 1. CROWLE, A. J. Immunodiffusion. Academic Press, New York, 1962, p Goss, D. A., and TAYMOR, M. L. Endocrinol. 71:321, OucHTERLONY, 0. Acta path et microbial. scandinav. 25:186, WIDE, L., and GEMZELL, C. Acta endocrinol. 89:539, 1962.