Despite the fact that a number of alterations of the hypothalamic-pituitary-gonadal hormone axis have been identified in patients with testicular cancer, little is known about the gonadotrophin secretion pattern in such patients who have greatly increased human chorionic gonadotrophin (hCG) serum concentrations. The aim of this study was to assess this issue in detail using a longitudinal study design and a panel of highly sensitive and specific immunoassays. Eleven patients with non-seminomatous (n=11), and one with seminomatous testicular cancer with pretreatment hCG serum concentrations exceeding 10(5) pg/ml (>1000 mIU/ml) were selected and followed for a mean of 166 days (mean of 14 serum samples/patient) after initial diagnosis. Serum concentrations of hCG, its free alpha- (hCGalpha) and beta- (hCGbeta) subunits, human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH) were determined by highly sensitive and specific enzymometric immunoassays based on a panel of monoclonal antibodies (MCA) established in our laboratory. A potential FSH-like activity (FSA) of hCG in the respective sera was determined by radioreceptor assays (RRA) for LH/CG and FSH. Specificity of FSA at the level of the receptor was assessed by MCA-based immunoabsorption studies. At diagnosis, hCG (9.8x10(7)+/-4.84x10(7) pg/ml; range 1.1x10(5)-5x10(8) pg/ml) was greatly increased and serum hFSH was undetectable (<9 pg/ml) in 11 patients, and one patient had very low, albeit detectable (approximately 30 pg/ml) hFSH concentrations. hLH was below the limit of detection (<2 pg/ml) in five individuals. During successful chemotherapy, hCG rapidly declined to physiological concentrations and hFSH/hLH returned to normal or even reached supraphysiological values. There was a highly significant negative correlation between hCG and hFSH (P=0.0001) and, to a lesser extent, hLH (P=0.0265). The ability of serum hCG to block the binding of [125I]rFSH (rat FSH) to its receptor was found to be 0.01-0.1% compared with the FSH standard; this could be reversed by an anti-hCG MCA. Addition of a specific MCA against hFSH blocked 3 microg/ml of the hFSH standard, but had no effect on the FSA of serum hCG in the FSH RRA. As observed during pregnancy, secretion of gonadotrophin -- particularly that of FSH -- is substantially or completely suppressed in patients with testicular cancer when serum hCG concentrations exceed 10(5)-10(6) pg/ml (approximately 10(3)-10(4) mIU/ml). As determined by RRA, the intrinsic FSA of tumour-derived hCG is most probably responsible for the suppression of hFSH in this group of patients with testicular cancer.

Abstract

Immunochemical studies were undertaken to identify surface-orientated epitopes of the free α subunit of human chorionic gonadotrophin (hCG-α) at the amino acid sequence level. We investigated the molecular organization of these epitopes, resolved the immunological topography in terms of spatial arrangement of antigenic domains and related structures to functions such as subunit association or receptor binding. Overlapping synthetic peptides covering the entire amino acid sequence of hCG-α, an enzymatically digested hCG-α subunit, and a reduced and alkylated hCG-α preparation were assayed in a solid-phase one-site enzyme-linked immunoassay, and in a solution-phase competitive radioimmunoassay (RIA). The antigenic topography was mapped by monoclonal antibodies (MCAs) in two-site binding assays (sandwich RIA). On the surface of hCG-α, seven different epitopes (α₁–α₇), arranged in four spatially distinct domains, could be distinguished: A, α_1,2,4; B, α_3,5; C, α₆; D, α₇. The peptides spanning hCG-α(13–18), hCG-α(17–22) and hCG-α(33–42) appeared to contribute to the formation of epitopes α₂, α₄ and α₆ respectively. Since epitope α₆ is present only on the free non-assembled subunit of different species, we concluded that the region hCG-α(33–42), which is evolutionarily highly conserved, represents a subunit assembly site. All but one epitope (α₇) are destroyed by reducing and alkylating hCG-α. In contrast, chymotryptic digestion of hCG-α, leading to release of the heptapeptide hCG-α(41–47), did not affect epitope expression, indicating that this sequence is not involved in the formation of antigenic determinants. Addressing the biological properties of hCG-α epitopes by radioreceptor assay revealed that the three hCG-α peptides corresponding to epitopes α₂, α₄ and α₆ did not displace radiolabelled hCG from its receptor, whereas any of the MCAs directed against determinants (α₁–α₅), shared by hCG and hCG-α, totally inhibited binding. Consistent with this, the antibodies neutralized the biological activity of hCG in terms of testosterone production in a mouse Leydig cell in vitro bioassay. We therefore concluded that hormone antibody-binding sites differ from those of hormone receptor binding, revealing no essential congruence of immunologically and biologically active domains.

Journal of Endocrinology (1994) 140, 145–154

ABSTRACT

Discordant results on body fluid levels of human chorionic gonadotrophin (hCG) free α- and β-subunits under physiological and pathophysiological conditions, prompted us to raise a total of 260 monoclonal antibodies (MCA) against free hCG-α, free hCG-β, holo-hCG, human follicle-stimulating hormone and bovine luteinizing hormone; 153 MCA recognizing the human α-subunit and 28 reacting with hCG-β were extensively analysed for their intra- and interspecies cross-reactivity with homologous hormones, and for the compatibility of epitopes recognized by them. The immunological topography of free hCG-α and free hCG-β was resolved by these MCA, and epitope maps were designed. Six antigenic determinants on the free α-chain (α₁–α₆), clustered in three spatially distinct domains, and seven epitopes on the surface of free hCG-β (β₁–β₇), could be distinguished. Strikingly, three α-chain epitopes (α₄, α₅ and α₆) were shared between various species, which is in contradiction to the concept of immunological species-specificity of α-subunits. Three determinants were found to be present only on the free subunits but not on holo-hCG (α₆, β₆ and β₇), and only two determinants (β₁ and β₇) were hormone-specific for hCG. Based on this information, an immunoenzymometric assay for the free α-subunit of human glycoprotein hormones was established, with a sensitivity of 1·3 pg/well and a cross-reactivity with holo-hCG of less than 0·005% Thus this assay provides the basis for detecting free α-subunits in the presence of extremely high levels of holo-hormones, which may assist in elucidating the role of free α-subunits in man.

Journal of Endocrinology (1990) 125, 301–309

Abstract

The molecular basis for antigenic determinants on the free β-subunit of human chorionic gonadotrophin (hCGβ), its carboxyl-terminal peptide (hCGβCTP) and the hCGβcore fragment (hCGβcf) was elucidated by means of monoclonal antibodies (MCAs). The objective of the present study was to resolve the antigenic topography of these three molecules in terms of epitope identification at different levels of structural organization as well as analysis of their spatial arrangement. An hCGβcf preparation, a synthetic peptide corresponding to the hCGβCTP (β109–145), overlapping synthetic peptides spanning the entire amino acid sequence of hCGβ, and a reduced and alkylated hCGβ preparation were assayed in a solid-phase one-site enzyme-linked immunoassay and in a solublephase direct-binding radioimmunoassay (RIA) or competitive RIA. The antigenic topography was mapped by incorporating the MCAs into two-site binding assays. On the surface of free hCGβ, nine different epitopes (β₁–β₉), arranged in three spatially distinct domains, could be distinguished. Epitopes β₁–β₇ were located in a single large domain on both hCGβ and the hCGβcf whereas hCGβCTP contained two topographically distant determinants, designated β₈ and β₉ respectively. All but the two epitopes located on hCGβCTP (β₈ and β₉) were destroyed by reducing and alkylating hCGβ, suggesting that most antigenic determinants are predominantly non-contiguous and require an intact tertiary structure whereas the molecular structure of hCGβCTP is linear. At a molecular level, amino acid residues spanning hCGβ 45–52, hCGβ 137–144 and hCGβ 113–116 contributed to the formation of epitopes β₅, β₈ and β₉ respectively. We have also shown that the hCGβcf represents the immunodominant part of the free β-subunit of hCG, containing seven mainly conformationally determined epitopes, one of which has a share of the sequence β45–52. The hCGβCTP does not play a critical role in the immunologically important tertiary structure of hCGβ and was itself found to be a predominantly continuous sequence also within the native hormone, expressing two spatially distant antigenic determinants located within residues β113–116 and β137–144 respectively.

Journal of Endocrinology (1994) 141, 153–162