Radioimmunoassay of Serum LH and Testosterone in

Radioimmunoassay of Serum LH and Testosterone in Male Rabbits Actively Immunized Against Testosterone1 IAN H. T H O R N E Y C R O F T , NANCY K. T H O R N E Y C R O F T , REX. J. SCARAMUZZI, 2 AND CHARLES A. BLAKE Department of Obstetrics and Gynecology, Section of Reproductive Biology, University of Southern California School of Medicine, Los Angeles, California 90033; Department of Anatomy, UCLA School of Medicine, Los Angeles, California 90024; and Department of Anatomy, Duke University School of Medicine, Durham, North Carolina 27710 ABSTRACT. Five male rabbits were actively immunized against testosterone to determine if this procedure could permanently inactivate circulating testosterone. To assess the response to immunization, serum LH, serum testosterone and antitestosterone titer (titer) were measured by radioimmunoassay at various times after immunization. All rabbits produced antisera to testosterone and developed serum LH levels similar to those measured in castrated males. The rise in serum LH was particularly pronounced after a booster immunization but these elevated levels were not maintained

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T has been demonstrated in castrated male rats administered with exogenous testosterone that the biological activity of testosterone can be neutralized by concurrent passive immunization against testosterone (1). Passive immunization is a suitable technique for short-term investigations but would be of no use in long-term experiments as the animals might develop antibodies to the administered antisera and could go into anaphylactic shock. Active immunization would presumably be the method of choice if a long-term or permanent effect was desired. In a preliminary short-term study by Thorneycroft (2) serum LH was shown to rise Received June 19, 1974. ' Supported by grants from the Ford Foundation, NIH (HSAA 5 SO4 RR06148), NIH (HD 01177) and the American Medical Association Education and Research Foundation. 2 Present address: Department of Obstetrics and Gynecology, University of Edinburgh, Edinburgh, U.K. EH3 9ER. Send reprint requests to: Dr. Ian H. Thorneycroft, Section of Reproductive Biology, Department of Obstetrics and Gynecology, University of Southern California School of Medicine, Women's Hospital, 1240 N. Mission Rd., Los Angeles, California 90033.

which suggests that biological neutralization was only transient. Serum testosterone rose dramatically after immunization and was directly correlated with titer. High serum testosterone concentrations were associated with both high and low serum LH. No correlation existed between titer and serum LH. It is concluded that active immunization against testosterone does not necessarily result in a permanent neutralization of circulating testosterone and that titer alone is an inadequate criterion of neutralization. (Endocrinology 97: 301, 1975)

after male rabbits were immunized against testosterone. Circulating testosterone was presumably biologically neutralized by immunization. Similar results have been reported by Hillier et al. (3) in rats. The present experiments were undertaken to investigate if active immunization against testosterone results in a relatively permanent or long-term neutralization of testosterone activity. The negative feedback relationship between testosterone and LH in males was used as an index of biological neutralization. Materials and Methods i. Animals. All rabbits used in this study were normal male New Zealand Whites obtained from a local breeder in California. ii. Immunization. All animals were immunized with a T-3-BSA conjugate (testosterone-3-0carboxymethyloxime-Bovine Serum Albumin) synthesized as previously described (4). One mg or 200 /xg of conjugate was dissolved in 0.5 ml of saline and then emulsified with 0.5 ml of Freund's complete adjuvant. Rabbits in group 1 (R6, R7 and R8) were given 1.0 mg of the conjugate in 5 dorsal subcutaneous sites weekly for 6 weeks; booster doses were ad-

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iii. Assays. Blood samples were drawn from the median ear artery before the initial immunization and at various times thereafter (see Figs. 1-3; rabbits R7 and R8 were bled the same as R6 until death). Samples were allowed to clot at room temperature and centrifuged to separate serum from cells.

ministered at various intervals thereafter (see Fig. 1 for R6). R7 and R8 received two booster doses at monthly intervals. Rabbits in group 2 (R21 and R22) were immunized by a modification of the method of Vaitukaitis et al. (5). They were given 200 /xg of conjugate in 5 dorsal subcutaneous sites and 0.5 ml of crude pertussis vaccine (53.8 B/cc, Lot No. BPO 695, Eli Lilly Co., Indianapolis, Ind.) in another subcutaneous site once at the beginning of the experiment. Both animals were boosted with 200 jxg of T-3-BSA 193 days after the initial immunization.

a) Titer: The titer of the antiserum was determined using a dextran-coated charcoal radioimmunoassay (6), and is defined as that dilution of antiserum which bound 50% of 10,000 dpm (40 pg) of tritiated testosterone

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FIG. 1. Antibody titer, serum LH and serum testosterone in rabbit R6 after immunization against testosterone. Dashed line ( ) indicates the upper limit of normal male serum levels of testosterone (13 ng/ml) and LH (37 ng/ml in terms of WP-360B). WP-360-B is approximately 10 times less potent than EX86AS used for R21 and R22.

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ACTIVE IMMUNIZATION AGAINST TESTOSTERONE (New England Nuclear; 45 Ci/mmol). A significant titer is arbitrarily defined in this study as anything greater than a 1:100 dilution.

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500 b) Testosterone: Serum testosterone was assayed by a radioimmunoassay method previously published (6) which uses celite column chromatography and dextran-coated charcoal. The following modifications were used: 0.1 ml of antiserum was diluted with 0.9 ml of water and 1000 dpm of tritiated testosterone (4 pg) was added to estimate recovery. Prior to extraction the recovery counts were incubated at 4 C for 16 h to allow full equilibration with the endogenous testosterone pool, most of which had been determined in preliminary experiments to be antibody bound. This modification was necessary as shorter incubations without sample dilutions resulted in an underestimation of antibody bound testosterone. After chromatography, 2 and 20% of the purified fraction werf taken for assay and 40% for recovery counting. A pool of normal male rabbit serum was run with each assay for quality control.

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200 c) LH: Serum LH was assayed using a heterologous double antibody radioimmunoassay specific for rabbit LH (7). A pool of male rabbit serum was run with each assay for quality control. Values for group 1 are expressed in terms of WP-360-B (0.025 x NIH-S1) and values for group 2 are expressed in terms of EX86AS (0.2 x NIH-S1).

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FIG. 2. Antibody titer, serum LH and serum testosterone in rabbit R21 before and after immunization against testosterone. Dashed line ( ) indicates the upper limit of the normal serum levels of testosterone and LH (4.8 ng/ml in terms of EX86AS).

All animals immunized produced antibody titers which, once elevated, remained at a greater than 1:100 dilution for the duration of the experiment or until death. Booster immunizations were followed by increased titers in both groups. Significant titers (> 1:100) were not observed before 6 weeks postimmunization, although the sampling was not frequent enough to state this categorically in rabbits R21 and R22. Serum testosterone concentration rose markedly after immunization and remained above preimmunization values throughout the experiment. The mean testosterone concentration of all samples after the initial immunization was 153 ng/ml ± 27.3 (mean ± SE) compared to 4.3 ± 0.84 ng/ml

in 16 normal nonimmunized male rabbits (P < 0.01). Patterns of total serum testosterone and titer were correlated [r = 0.88 with 33 degrees of freedom (df); P < 0.01]. The mean serum LH concentration in 8 castrated male rabbits 1 to 2 weeks after castration was 12.6 ± 1.1 ng/ml (mean ± SE) in terms of the EX86AS standard. In Figs. 1-3 the titer, serum LH and serum testosterone values for rabbits R6, R21, and R22 are shown respectively. Rabbit R6 (Fig. 1) had elevated serum LH at 3.0 months which rose further to castration levels at 4 months and remained at elevated levels until 4.7 months. Serum LH then declined to normal levels which were maintained until the animal was



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boosted again at 16.3 months. Serum LH concentration then rose to castration levels for a second time but did not remain elevated. Rabbit R21 had elevated LH levels at 1.5, 3.0 and 4.2 months after the first immunization, and normal levels at all other times until the booster injection at 6.4 months. After the booster, the LH levels rose to castration levels within one month and then declined. Rabbit R22 had castration LH

values at 1.9 and 4.2 months and normal levels at all other times including after the booster injection at 6.4 months. Serum LH concentration in R6 and R22 (Figs. 1 and 3) did not rise until after the elevation in both serum testosterone and titer. However, in R21 all three showed an elevation at the same time (Fig. 2). The coefficient of correlation between titer and serum LH concentration was 0.23 with 45 df which is not statistically significant


ACTIVE IMMUNIZATION AGAINST TESTOSTERONE

(P > 0.05). Titers as high as 1:6000 were associated with normal serum LH levels (Fig. 1). Nonelevated LH levels were associated with serum testosterone concentrations ranging between 2.3 and 803 ng/ml. The coefficient of correlation between serum testosterone and LH was 0.35 with 48 df (0.01 < P < 0.05). Rabbits R7 and R8 died of unknown causes before the experiment was terminated. Data obtained from them are included in the correlations and mean data. The titers in R7 and R8 were lower than those observed in R6 (1:200-1:2000). Discussion Passive immunization provides a very useful and specific tool for short-term neutralization of a given hormone (8-12). The studies of Scaramuzzi et al. (13) and Caldwell et al. (11,14) demonstrated the potential use of active immunization to specifically neutralize endogenous steroid levels over longer periods of time. In the latter studies of Caldwell et al. (14) no reliable criterion or index of neutralization of estradiol such as LH or ovarian weight was measured. Thorneycroft (2) and Hillier et al. (3) indicated that active immunization against testosterone could indeed neutralize serum testosterone in male rabbits and rats as evidenced by elevated serum LH. These experiments were over relatively shorter periods than the results presented in the present study. All animals produced antibodies, but the serum LH was not necessarily elevated. Serum LH did not rise parallel with the anti-testosterone titers but rose later in two of the three closely monitored animals. The highest antibody titers were not always associated with the highest serum LH concentrations. Rabbit R6 was not boosted for a 9-month period during which serum LH concentrations were normal and the antitestosterone titers had plateaued at ca. 1:700 to 1:1000 (Fig. 1). Titer is therefore not a good index of neutralization.

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Serum testosterone was greatly elevated in all immunized animals, which is consistent with previous reports (3,15). The highest levels observed were 200-fold higher than normal but were associated with either normal or greatly elevated serum LH concentrations. There was a significant but low positive correlation between the serum testosterone and LH levels. We place no significance on this correlation as there is no apparent trend when the individual values are examined. In animals R6 and R22, serum testosterone levels rose prior to any rise in serum LH. These results differ from those reported in the rat by Hillier et al. (3) where the serum LH rose with serum testosterone and before the rise in titer. In addition, the serum LH levels reported by Hillier et al. (3) remained elevated for the duration of the experiment (16 weeks). Plasma LH concentration fluctuates in a pulsatile rhythm in long-term ovariectomized rabbits (Scaramuzzi, Scaramuzzi and Hilliard, unpublished). It is possible that plasma LH fluctuates in this manner after immunization and that some of the low LH values may be due to sampling during periods of low LH release. Such a phenomenon could account for the low LH observed at 1.9 months in R21 and at 3.0 months in R22. However, such an explanation probably cannot account for all of the low values as LH is consistently lowered at times; for example, 4.8 months to 16.3 months in R6 and from 5.5 months on in R22. In examining the individual time courses in rabbits R6, R21 and R22 it is concluded that as the antibody titers rose, testosterone apparently became bound to the antibody and elevated testosterone levels resulted. This latter conclusion gains support from the fact that the serum testosterone and the antiserum titer were significantly correlated. In many cases, elevated testosterone levels were associated with elevated serum LH concentrations, rather than with low serum LH concentrations, as might have been anticipated in a negative feedback relation-


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These experiments demonstrate that any ship. In these cases, testosterone bound to the antibody must have become biologically endocrine system which has a negative neutralized relative to the hypothalamic- feedback may be difficult to alter either hypophyseal axis. As the titers rose, enough for long periods of time or permanently by of the testosterone was neutralized to result active immunization. This conclusion would in increased serum LH. The high serum also hold true for passive immunization LH levels would presumably have caused studies. increased testosterone production by the After completion of this manuscript, a testes. It is known that binding proteins paper by Ferin et al. (17) appeared in which in blood decrease the metabolic clearance monkeys were actively immunized against rate of steroids (16); therefore, the in- estradiol and results similar to those recreased production rate of testosterone ported here were observed. combined with its lower clearance rate References would result in higher serum testosterone 1. Neri, R. O., S. Tolksdorf, S. M. Beiser, B. F. levels. It appears in rabbits R6, R21 and R22 that this neutralization of circulating

testosterone by the antisera eventually ceased even though there were antitestosterone titers. Perhaps at this point all of the antibody combining sites had been saturated or saturated sufficiently enough, so that the non-antibody bound testosterone reached normal levels and serum LH concentration returned to normal. Testosterone levels as low as 0.5 ng per ml have been measured in the serum of the normal non-immunized rabbit and these levels maintain a normal negative feedback relationship with serum LH. Therefore, it would require that less than 0.5% of the total serum testosterone in actively immunized animals be non-antibody bound in order to maintain normal LH levels. The question of how much is bound and how much is non-antibody bound could possibly be ascertained by equilibrium dialysis studies. We did not perform equilibrium dialysis experiments as they result in dilution of the serum, and it would be difficult to draw any conclusions regarding the amount of testosterone bound in undiluted serum from such experiments. Hillier et at. (3) did perform equilibrium dialysis and concluded that during early immunization very little testosterone was non-antibody bound, and that the quantity increased with time, but never reached normal levels. In their experiments the antiserum was diluted 1:10 and then dialyzed against 10 x volume.

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Erlanger, F. S. Agate, Jr., and S. Lieberman, Endocrinology 74: 592, 1964. Thomeycroft, I. H., Biol Reprod 7: 116, 1972. Hillier, S. G., E. N. Cole, G. V. Groom, A. R. Boyns, and E. H. D. Cameron, Steroids 22: 227, 1973. Erlanger, B. F., F. Borek, S. M. Beiser, and S. Lieberman, J Biol Chem 228: 713, 1957. Vaitukaitis, J., J. B. Robbins, E. Nieschlag, and G. T. Ross, J Clin Endocrinol Metab 33: 988, 1971. Barberia, J. M., and I. H. Thomeycroft, Steroids 23: 757, 1974. Scaramuzzi, R. J., C. A. Blake, H. Papkoff, J. Hilliard, and C. H. Sawyer, Endocrinology 90: 1285, 1972. Goodfriend, L., and A. H. Sehon, Can J Biochem Physiol 39: 962, 1961. Ferin, M., P. E. Zimmering, S. Lieberman, and R. L. Vande Wiele, Endocrinology 83: 565, 1968. , J. Raziano, A. Tempone, and R. L. Vande Wiele, In Peron, F. G., and B. V. Caldwell (eds.), Immunologic Methods in Steroid Determination, Appleton Century-Crofts, New York, 1970, p. 199. Caldwell, B. V., R. J. Scaramuzzi, S. A. Tillson, and I. H. Thomeycroft, ibid., p. 196. Raziano, J., M. Ferin, and R. L. Vande Wiele, Endocrinology 90: 1133, 1972. Scaramuzzi, R. J., S. A. Tillson, I. H. Thomeycroft, and B. V. Caldwell, Endocrinology 88: 1184, 1971. Caldwell, B. V., S. A. Tillson, H. Esber, and I. H. Thomeycroft, Nature (Lond) 231: 118, 1971. Nieschlag, E., K. Usadel, U. Schwedes, H. K. Kley, K. Schoffling, and H. L. Kriiskemper, Endocrinology 92: 1142, 1973. Baird, D. T., R. Horton, C. Longcope, and J. F. Tait, Recent Progr Horm Res 25: 611, 1969. Ferin, M., I. Dyrenfurth, S. Cowchock, M. Warren, and R. L. Vande Wiele, Endocrinology 94: 765, 1974.
