title: Production of a monoclonal antibody to cortisol: application to a direct enzyme-linked immunosorbent assay of plasma.
abstract: Cortisol mouse monoclonal antibodies were produced and characterized. Of the four clones studied, supernatant from one clone (A2), compared with other cortisol monoclonal antibodies, showed minimal cross-reactivity to other C21 steroids and was suitable for the direct determination of cortisol in plasma by enzyme-linked immunosorbent assay using a standard 96-well microtiter plate. The enzyme-linked immunosorbent assay uses the immobilized antigen approach, in which cortisol in plasma samples or standards competes with immobilized steroid for antibody-binding sites. After washing, the cortisol antibody bound to the wells of the microtiter plate is detected with antimouse immunoglobulin conjugated to horseradish peroxidase. Following further washing, o-phenylenediamine substrate is added. The enzyme-linked immunosorbent assay is robust and semiautomated. The mean +/- SD recovery from plasma was 97% +/- 6%. Precision studies on three different plasma pools showed mean coefficients of variation of 7.6% and 8.6% for within- and between-assay variation, respectively. The satisfactory performance criteria allow its use in the routine laboratory.
authors.affiliation: Department of Clinical Biochemistry, Christchurch Hospital, New Zealand.
authors.forename: J G
authors.initials: JG
authors.lastname: Lewis
authors.forename: L
authors.initials: L
authors.lastname: Manley
authors.forename: J C
authors.initials: JC
authors.lastname: Whitlow
authors.forename: P A
authors.initials: PA
authors.lastname: Elder
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006854:Hydrocortisone / Q000097:blood* / Q000276:immunology; D008797:Metyrapone / Q000494:pharmacology; D051379:Mice; D013256:Steroids / Q000097:blood / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D013256:Steroids; D006854:Hydrocortisone; D008797:Metyrapone