Candidate epitopes for measurement of hCG and related molecules: the second ISOBM TD-7 workshop

Participants of the Second International Workshop (WS) on human chorionic gonadotropin (hCG) of the International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) have characterized in detail a panel of 69 antibodies (Abs) directed against hCG and hCG-related variants that were submitted by eight companies and research groups. Specificities of the Abs were determined using the First WHO International Reference Reagents for six hCG variants, i.e., hCG, hCGn, hCG beta, hCG beta n, hCG beta cf, and hCG alpha, which are calibrated in SI units, and hLH. Molecular epitope localizations were assigned to the ISOBM-mAbs by comparing ISOBM-Ab specificity, sandwich compatibility, and mutual inhibition profiles, to those of 17 reference monoclonal (m)Abs of known molecular epitope specificities. It appeared that 48 Abs recognized hCG beta-, 8 hCG alpha-, and 13 alpha beta-heterodimer-specific epitopes. Twenty-seven mAbs were of pan hCG specificity, two thereof with no (< 0.1 %; epitope beta(1)), 12 with low (< 1.0 %; epitopes beta(2/4)), and 13 with high (>> 1 %; epitopes beta(3/5)) hLH cross-reactivity. The majority of hCG beta epitopes recognized were located in two major antigenic domains, one on the peptide chain of the tips of beta-sheet loops 1 and 3 (epitopes beta(2-6); 27 mAbs) and the second around the cystine knot (e.g., epitopes beta(1), beta(7), and beta(10); 9 mAbs). Four mAbs recognized epitopes on hCG beta cf-only (e.g., epitopes beta(11) and beta(13)) and six mAbs epitopes on the remote hCG beta-carboxyl-terminal peptide (epitopes beta(8) and beta(9) corresponding to amino acids 135-144 and 111-116, respectively). For routine diagnostic measurements, methods are used that either detect hCG-only, hCG beta-only, or hCG together with hCG beta or hCG together with hCG beta and hCG beta cf. Sandwich assays that measure hCG plus hCG beta and eventually hCG beta cf should recognize the protein backbone of the analytes preferably on an equimolar basis, should not cross-react with hLH and not be susceptible to blunting of signal by nonmeasured variants like hCG beta cf. Such assays can be constructed using pairs of mAbs directed against the cystine knot-associated epitope beta(1) (Asp10, Asp60, and Gln89) in combination with epitopes beta(2) or beta(4) located at the top of beta-sheet loops 1 + 3 of hCG beta involving aa hCG beta 20-25 + 68-77. In summary, the results of the First and Second ISOBM TD-7 WSs on hCG provide the basis for harmonization of specificities and epitopes of mAbs to be used in multifunctional and selective diagnostic hCG methods for different clinical purposes.