Monoclonal antibodies to human sex hormone-binding globulin (SHBG): Characterization and use in a simple enzyme-linked immunosorbent assay (ELISA) ofSHBG in plasma

Four monoclonal antibodies re, human sex hormone-binding globulin were rais ed and characterized. Three of the four antibodies recognised different ant igenic determinants on SHBG. Two of the distinct antibodies were useful for Western blotting and recognized a major 48 kDa band in human plasma as wel l as a 46 kDa minor component. Carbohydrate residues do not form part of th e antigenic determinants of these two antibodies, although one of these sho wed increased signal following removal of N-linked oligosaccharides. Some o f the antibodies were selected to form a basis of a same-day, non-competiti ve, enzyme-linked immunosorbent assay (ELISA) for SHBG in plasma. The assay employs a purified IgG2a SHBG monoclonal antibody adsorbed to the wells of a microtitre plate. After blocking any further adsorption to the plate, st andards or diluted patient samples were added for a 5-h incubation at room temperature, after which the plate was washed and antibody-bound SHBG was d etected with an anti-SHBG IgG1 monoclonal antibody followed by peroxidase-l abeled antimouse-IgG1 and o-phenylenediamine substrate. The assay correlate d well with an existing 2-day ELISA for SHBG in plasma using polyclonal ant ibodies and also correlated with a dihydrosterone (DHT) ligand-binding assa y. The monoclonal antibody-based ELISA shows excellent performance characte ristics and is unaffected by added testosterone or estradiol, (C) 1999 Else vier Science Inc. All rights reserved.