EUROPEAN COLLABORATIVE STUDY OF LH ASSAY .3. RELATIONSHIP OF IMMUNOLOGICAL REACTIVITY, BIOLOGICAL-ACTIVITY AND CHARGE OF HUMAN LUTEINIZING-HORMONE

This report describes the results of the third part of the collaborati ve study organized by a working group sponsored by the Community Burea u of Reference of the European Community Commission. The aim of the pr esent work was to establish the link between immunoreactivity and biol ogical activity of human LH, thus allowing to determine the antigenic domains of the molecule involved in the induction of the biological ef fect. The relationship between immunoreactivity and electric charge of hLH was also studied. This work allowed to further apprehend hLH isom orphism and its role in discrepancies observed among hLH assays and cl inical status, It also made the feasibility of measuring biologically active isoforms by an immunological method to be assessed, The effect of 36 mAb with known epitopic specificity, was evaluated on both hLH b inding to rat membrane receptor and hLH induced production of testoste rone by porcine Leydig cells, All the epitopes located on the beta sub unit were found to be strongly involved in the biological activity whe reas 4/9 and 10/18 epitopes present on the ex subunit or specific for the holomolecule respectively appeared weakly involved. Assaying biolo gical hLH using immunological method would require that mAb specific f or all the epitopes involved in the receptor activation be tested, and thus appears presently unsuitable for routine clinical evaluation. In the previous work some LH immunoassays were found to underestimate LH concentrations (J. Endocrinol. Invest 1994, 17: 397-406 and 407-416). The mAb used in liquid phase in these kits were found in the present work to be directed against the domains of LH weakly involved in the a ctivation of the receptor and would suggest that bioactive LH would be misevaluated by these kits. The immunoreactivity of hLH isoforms sepa rated by isoelectric focusing (IEF) in liquid phase was also determine d. IEF allowed to separate three groups of hLH isoforms but none of th em exhibited a specific discriminating pattern of immunoreactivity whe n they were tested against a panel of mAb. It suggests that, in our ex perimental conditions, the electric charge and the immunoreactivity of hLH were not closely linked.