An improved solid-phase enzyme and luminescent immunoassay system for steroid hormones and digoxin

Abstract

A uniform solid-phase system has been developed for enzyme (ELISA) or luminescent (LIA) immunoassays for steroids. These assays were improved by (a) irradiating microtiter plates or polystyrene tubes before coating with antibody or Protein A, (b) coating the plastic trays with nonspecific anti-gamma-globulin or Protein A instead of the steroid-specific first antibody, and (c) partial denaturation of the second antibody before coating the plates or tubes with it. Specific antibodies were raised against cortisol, aldosterone, 17-hydroxyprogesterone, and digoxin. Horseradish peroxidase was used as label for the ELISA and aminoethylisoluminol for the LIA. In comparison with the first (specific) antibody coating method we observed some advantages: From 10- to 33-fold lower concentrations of first antibodies were necessary to bind the same amount of steroids; precision was better (CV, 3.8-7.5% vs 6.9-15.5%). The high sensitivity of these assays (0.5-2.0 pg per tube for the steroids) also allows determination of the steroids and digoxin in plasma and saliva.