Enzyme-Labelled Immunoassay for Plasma Cortisol

Abstract

An enzyme-labelled immunoassay for plasma cortisol was developed. For this alkaline-phosphatase was conjugated through 21-hemisuccinate of cortisol using water-soluble carbodiimide. An antibody was raised in rabbits against corisol-21-hemisuccinate bovine serum albumin. Before assay 10 mul samples of plasma were mixed with glutamate buffer, pH 3.3, and boiled to denature endogenous cortisol-binding globulin and alkaline-phosphatase. Separation of "bound and free" cortisol was done by the double antibody method. A linear relationship was obtained between the volume of plasma and the amount of cortisol. The minimal detectable level of plasma cortisol was 1 mug/dl and the coefficients of variation were 2.7%-4.4% (within assays) and 4.7%-6.0% (between assays). Cortisol values determined by the present method correlated well with those determined by radioimmunoassay. The present method of enzyme-labelled immunoassay is suitable for routine clinical analysis of plasma cortisol.