Monoclonal antibodies to human sex hormone-binding globulin (SHBG): characterization and use in a simple enzyme-linked immunosorbent assay (ELISA) of SHBG in plasma.
Four monoclonal antibodies to human sex hormone-binding globulin were raised and characterized. Three of the four antibodies recognised different antigenic determinants on SHBG. Two of the distinct antibodies were useful for Western blotting and recognized a major 48 kDa band in human plasma as well as a 46 kDa minor component. Carbohydrate residues do not form part of the antigenic determinants of these two antibodies, although one of these showed increased signal following removal of N-linked oligosaccharides. Some of the antibodies were selected to form a basis of a same-day, non-competitive, 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, standards 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 detected with an anti-SHBG IgG1 monoclonal antibody followed by peroxidase-labeled antimouse-IgG1 and o-phenylenediamine substrate. The assay correlated well with an existing 2-day ELISA for SHBG in plasma using polyclonal antibodies and also correlated with a dihydrosterone (DHT) ligand-binding assay. The monoclonal antibody-based ELISA shows excellent performance characteristics and is unaffected by added testosterone or estradiol.
Lewis
J G
JG
Steroid & Immunobiochemistry Laboratory, Canterbury Health Laboratories, Christchurch, New Zealand. johnL2@chhlth.govt.nz
Longley
N J
NJ
Elder
P A
PA
D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000941:Antigens / Q000032:analysis / Q000276:immunology; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008297:Male; D051379:Mice; D011247:Pregnancy; D012016:Reference Values; D012738:Sex Hormone-Binding Globulin / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000941:Antigens; D012738:Sex Hormone-Binding Globulin