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We used immunological techniques to compare the serum corticosteroid-binding globulins (CBG) and testosterone-estradiol-binding globulins (TeBG) of Old World primates (man, chimpanzee, cynomologus, and rhesus), New World monkeys (squirrel and owl), and prosimians (galago and lemur). Four different antihuman TeBG antisera could not differentiate human and chimpanzee TeBG and recognized the galago and lemur TeBG as similar as well as the rhesus and cynomologus TeBG, as similar. Western blots of serum subjected to sodium dodecyl sulfate gel electrophoresis, with detection by an anti-TeBG antiserum, showed similar patterns of distribution of the two molecular species of TeBG for all of the New World primates and the owl monkey. The abundance of the two TeBG species was reversed in squirrel monkey serum, while lemur and galago displayed only a single band. Four different antihuman CBG antisera grouped together the CBGs of human and chimpanzee, rhesus and cynomologus, and lemur and galago. The squirrel monkey has a CBG with a markedly decreased affinity for cortisol; all four antisera perceived its CBG as much more immunologically distant from the human protein than that of the owl monkey. Indeed, three of the four antisera grouped squirrel monkey CBG with that of the prosimians, while one antiserum saw squirrel monkey CBG as even more distant from the human protein than the CBG of the primitive primates, the prosimians.

A series of four monoclonal antibodies to rat androgen-binding protein (ABP) has been developed. These antibodies recognize both the heavy (48,400-dalton) and light (43,000-dalton) subunits of the native ABP molecule. In addition, they recognize the subunits from which Asn-linked oligosaccharides have been removed by treatment with N-glycanase, indicating that these moieties do not form the immunological determinants recognized by the antibodies. Two of these antibodies are capable of recognizing both nondenatured ABP, as assessed by dot blot analysis, and denatured ABP, as determined by Western blot analysis of ABP after electrophoresis under denaturing conditions. The immunoreactivity of denatured ABP is decreased with two of the antibodies, suggesting that they more readily recognize the antigenic epitopes when the protein is in its native configuration. The antibodies were capable of immunoprecipitating covalently labeled ABP from solution. All four monoclonal antibodies produced were determined to be immunoglobulins M by both enzyme-linked immunosorbent assay and Ouchterlony immunodiffusion even though the initial serum response of the immunized animals indicated the presence of immunoglobulins G. All of the monoclonal antibodies raised against rat ABP cross-reacted with rabbit and human testosterone-binding globulin (TeBG). They were able to detect two subunits when Western blots of intact rabbit [mol wt (Mr, 43,000 and 40,500] or human (Mr, 47,600 and 44,500) TeBG were probed, but only a single subunit (Mr, 39,300) when deglycosylated samples of rabbit TeBG were analyzed.

Two monoclonal antibodies (MK1 and MK2) reacting with human sex-hormone binding globulin (SHBG) were obtained from mice hybridomas. The dissociation constants for the binding of SHBG to MK1 and MK2 were 7.5 and 75 pmol/L, respectively. MK1 was coupled to polyacrylamide beads with a yield of 37%, resulting in 15 mg of antibody per gram of beads. The maximal binding of SHBG by the MK1-beads was 16% of the theoretical capacity. The amount of 125l-labeled MK2 bound to MK1-beads was related to the amount of SHBG present. The system has been used for the immunoradiometric assay (IRMA) of SHBG in serum, and has been standardized with purified SHBG. Assay sensitivity is 3 micrograms/L; intra- and inter-assay (total variation) CVs were 5% and 10%, respectively. Values obtained with the assay for 100 patients' sera agreed well with those obtained with a conventional radioimmunoassay, and SHBG in a patient's serum subjected to gel chromatography eluted as a symmetrical peak with the expected retention when the effluent was analyzed with the present assay. Analytical recovery of SHBG added to serum from a man, a woman, and a pregnant woman ranged between 93% and 107%. The mean (and SD) concentrations of SHBG in sera from healthy women and men were 3.7 +/- 1.1 and 1.8 +/- 0.9 mg/L, respectively.

Antiserum against human testosterone-estradiol-binding globulin (hTeBG) was prepared in rabbits, and its specificity was demonstrated by crossed immunoelectrophoresis. A RIA for the measurement of hTeBG in serum was developed. With this assay, hTeBG was readily measured in 1-2 microliters serum. Interassay coefficients of variation for pools of serum from men, women, and women in late pregnancy were 7.7%, 5.5%, and 6.1%, respectively. Interassay coefficients of variation for the same pools were 10%, 12%, and 12%, respectively. The TeBG levels in a number of nonselected subjected determined by the present method show good correlations with those obtained by steady state polyacrylamide gel electrophoresis and dextran-coated charcoal assay. The concentrations of TeBG determined by the RIA in sera from men, women, and women in late pregnancy were 18 +/- 9 (n = 12), 54 +/- 13 (n = 8), and 374 +/- 55 (n = 6) pmol/ml (mean +/- SD), respectively.

In the human, there are two glycoproteins, testosterone-estradiol-binding globulin (hTeBG) and androgen-binding protein (hABP), which bind testosterone. Although these two proteins have similar physicochemical properties, they can be distinguished on the basis of origin and lectin binding. hTeBG is made in the liver and exhibits high affinity for Concanavalin A (Con A), while hABP from the testes is only partially bound to this lectin. That is, when testicular extracts were applied to Con A-Sepharose columns, a portion of the testosterone-binding material showed no interaction with the lectin and eluted in the void volume (peak I), while the remainder interacted strongly and could be eluted with alpha-methyl-D-glucoside (peak II). These observations are consistent with the proposal that peak I contains only hABP, whereas peak II contains hTeBG and/or hABP with carbohydrate units that permit binding to Con A. To further study the properties of these binding proteins, a hTeBG RIA using a monospecific antiserum was employed to compare the proteins in testes and serum. The results indicated that the testosterone-binding activities in peaks I and II of testicular extracts could not be distinguished immunologically from hTeBG in sera of normal women. These findings suggested that hTeBG and hABP share common epitopes. We next determined whether hABP was secreted into the blood or amniotic fluid by fractionating these fluids in Con A-Sepharose columns. Unlike testicular extracts, male serum and amniotic fluid contained single immunoreactive and steroid-binding species which bound specifically to Con A. We conclude from these observations that hABP (peak I), peak II activity, and hTeBG have common immunodeterminants and that if hABP is secreted into the blood of men, then its carbohydrate chains bind to Con A, making it indistinguishable from hTeBG under these conditions.