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Human growth hormone 20 kDa (20 kDa-hGH) is a natural variant of the main hGH isoform (22 kDa-hGH). Since some 20- and 22 kDa-hGH biological activities are not identical, we decided to map the prolactin (PRLR) and growth hormone receptor (GHR) binding sites for both isoforms. Monoclonal antibody (MAb) R7B4, directed to both receptors, was employed to estimate the relative proximity between 20- and 22 kDa-hGH receptors binding sites. Results indicated that although both hGH isoforms share the same PRLR present in Nb2-cells and rat liver, MAb R7B4 differently affected hormone binding, suggesting that their receptor binding sites would be close in Nb2-cells and separate in rat liver membranes. Since labelled 20 kDa-hGH did not bind significantly to hGHR, we added to the incubating medium an allosteric MAb anti-hGH that improved 20 kDa-hGH affinity for receptors. Under these experimental conditions MAb R7B4 inhibited 20 kDa-hGH binding to human liver but not to placenta, whereas the Ab impaired 22 kDa-hGH binding to both receptors. Data thus suggested that both hGH isoforms share the same hGHR binding site in liver tissue but bind to different overlapped regions in placenta. Consequently, results presented in this paper indicate that PRLR and GHR binding sites for 22- and 20 kDa-hGH should not be always identical, a fact that could explain some of the isoforms different activities.

An immunization protocol that induces antibodies (Abs) directed to cryptic epitopes of a protein antigen (Ag) reduces the efficacy of vaccines that ideally should induce Abs against native epitopes. We have shown earlier that viral infections concomitant with immunization against a protein tend to shift the Ab specificity toward cryptic epitopes and tend to induce the production of autoantibodies (autoAbs). Here, we show the effects of three adjuvants on the Ab specificity in the absence or presence of a viral infection (lactate dehydrogenase-elevating virus or LDV), with human growth hormone (hGH) being, as before, the protein Ag. Pathogen-free CBA/Ht and BALB/c mice were immunized with hGH in the presence of complete Freund's adjuvant (CFA), monophosphoryl lipid A (MPL) or alum, with the animals being either infected with LDV or not infected with LDV. Conventional and competition enzyme-linked immunosorbent assays (ELISAs) indicated that in noninfected mice, CFA induced higher titres of anti-hGH Ab than did MPL or alum, with the Ab being almost totally directed to cryptic hGH epitopes. Strikingly, CFA plus LDV infection in CBA/Ht mice shifted the specificity of the anti-hGH Ab toward native epitopes, whereas the virus decreased the Ab titre when MPL or alum was used. Our Western blot results showed that 70% of mice immunized with hGH in the presence of any adjuvant produced autoAbs against a variety of tissue Ags. The amount of autoAb and the concentration of Ab to hGH cryptic epitopes did correlate, suggesting a relationship between both kinds of Ab. Significant differences were observed in the various effects of adjuvants and the viral infection between the two mouse strains used in this work.

Anti-human growth hormone (hGH) polyclonal and monoclonal antibodies (MAb) failed to recognize ovine placental lactogen (oPL), indicating that the antigenic topographies of both hormones are different. Binding assays showed that oPL completely inhibited hGH binding to lactogenic receptors from Nb2-cells and to somatogenic receptors from rabbit or sheep liver; in contrast, oPL only bound to a subpopulation of rat liver receptors. Zinc ion increased hGH and oPL binding to Nb2-cell receptors and slightly inhibited both hormones' recognition by somatogenic receptors. However, ZnCl(2) increased hGH binding to rat liver microsomes but prevented that of oPL. Furthermore, MAb R7B4, recognizing lactogenic as well as somatogenic receptors, entirely blocked hGH binding to the various receptor systems but not affected oPL binding. Therefore, results presented in this paper suggest that oPL and hGH bind to different regions of the same receptors., (Copyright 1999 Harcourt Publishers Ltd.)