AN ELECTROCHEMICAL ENZYME-IMMUNOASSAY FOR CHICKEN LUTEINIZING-HORMONE- EXTENSION OF THE DETECTION LIMIT BY ADEQUATE CONTROL OF THE NONSPECIFIC ADSORPTION

A noncompetitive heterogeneous enzyme immunoassay for the determinatio n of chicken luteinizing hormone (LH) was equipped with an electrochem ical endpoint in order to further enhance its sensitivity. The immunol ogical principle of the original ELISA remained essentially unchanged, except for the fact that the peroxidase label was replaced by alkalin e phosphatase, since in the upgraded version of the assay, p-aminophen yl phosphate was to be used as the substrate of alkaline phosphatase. Enzyme-generated p-aminophenol was injected into a how-injection syste m and detected amperometrically in a thin-layer how cell with a glassy carbon electrode at 0.325 V vs Ag/ AgCl, A classical problem associat ed with this type of solid-phase immunoassay is the adsorption of prot eins other than the capture antibody to the solid phase. The detection sensitivity is therefore often limited by a large background signal o bserved in the absence of antigen. In the present study, an experiment was designed to examine in each step of the assay the contribution of each of the potential sources of background current. It was shown tha t the major contribution to the background current was caused by the n onspecific adsorption of biotinylated secondary antibody. Adsorption o f the secondary antibody (biotinylated goat anti-rabbit IgG) to the ca pture antibody (mouse anti-chicken LH beta) was clearly a case of spec ific aspecificity, whereas adsorption to the solid phase itself had to be treated as a nonspecific aspecificity. Addition of 0.25% mouse ser um to the secondary antibody as a source of mouse immunoglobulin could overcome the cross-reaction and markedly reduced adsorption to captur e antibody. The second part of nonspecific adsorption was eliminated b y using combinations of Tween 20 and bovine serum albumin as blocking agents. Controlling the adsorption of the biotinylated secondary antib ody in this way decreased the detection limit from 39 pg/ml in the ori ginal assay to 2.5 pg/ml in the electrochemical version. This way, the plasma volume of samples containing on the order of 1 ng/ml LH was re duced to less than 10 mu l. The linear range was 2.5-625 pg/ml, The me thod allowed us to measure LH in buffer and in adult and juvenile chic ken plasma. (C) 1998 Academic Press.