Characterization Of A Monoclonal Antibody Reacting With The Free Human Luteinizing Hormone Beta-Subunit

Measurement of serum luteinizing hormone (hLH) is important for the detection and follow-up of patients with pathological processes of the reproductive axis. Detection of the uncombined form of the beta-subunit of hLH, or free hLH beta, has proved to be of clinical interest in the recognition of gonadotroph adenomas. As no monoclonal antibody specific for the free hLH beta is at present available, we elicited monoclonal antibodies using free hLH beta as an immunogen. An antibody, named BLH01, was selected for its specific binding to the free beta-subunit, and its antibody-binding site was characterized at the molecular level, emphasizing the importance of amino acid residues located between the disulfide-bonded Cys(93) and Cys(100). This region has been demonstrated as being particularly critical for the specific binding of lutropic hormones to their receptors. Topographic assignment of the epitope recognized by BLH01 was then achieved by crossmatching studies based on a library of antibodies directed to hLH beta, and the location of some epitopes on the three-dimensional model of the beta-subunit is proposed. A two-site immunoassay based on BLH01 as capture antibody was then developed. This assay, using BLH01, may constitute a simple, sensitive and highly specific procedure for assessing the clinical usefulness of measuring free hLH beta, particularly for the diagnosis and follow-up of patients presenting with pituitary adenomas.