ENZYME-AMPLIFIED IMMUNOASSAY FOR STEROIDS IN BIOSAMPLES AT LOW PICOMOLAR CONCENTRATIONS

A competitive enzyme amplified ELISA for steroids was developed using recycling of NADH/NAD+ between the enzymes diaphorase and alcohol dehy drogenase. The substrate was generated from the steroid-bound enzyme l abel alkaline phosphatase, which dephosphorylated NADPH or NADP+. Seco ndary antibodies were partially denatured and adsorbed to the microtit re plates to overcome the inhomogeneity of the plastic material. In th e amplified ELISA reported here, amounts down to 1 femtomol per well o f the steroid budesonide could be quantified with a relative standard deviation of 30%. Plasma samples were pretreated using a solid phase e xtraction and a subsequent column liquid chromatography fractionation. Concentrations in blood plasma could be quantified down to 8 pM (5 fm ol per well) with a precision of better than 20%. Different detection principles for the alkaline phosphatase label were compared. and the p roposed double amplification procedure was found to give a substantial increase in detectability of the enzyme conjugate compared to the con ventional detection of p-nitrophenol.