Analytical performance of luminescent immunoassays of different format for serum daidzein analysis

Two sensitive competitive-type solid-phase immunoassays for serum daidzein analysis have been developed and optimized. The first is a chemiluminescent enzyme immunoassay that uses black polystyrene microtiter wells in which daidzein-specific antibodies raised in rabbits are immobilized and a daidzein derivative is coupled to horseradish peroxidase (HRP) as a label. The HRP activity of the antibody-bound tracer is measured with an enhanced chemiluminescent system (luminol/ H₂O₂/enhancer). The second immunoassay is based on the use of bovine serum albumin–daidzein derivative immobilized on microtiter plates and a secondary anti-rabbit IgG-Fc fragment conjugated with 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Formation of the complex Eu³⁺-BCPDA enables time-resolved fluorescence-mode detection of the amount of antibody bound to the immobilized antigen. Both methods fulfilled all the requirements of accuracy and precision. The detection limit was the same for each method, 10 pg/ well; this is better than that of other immunoassays. The specificity of the two methods was different, because of their competitive-type mechanisms. The performance of the chemiluminescence method is better, because the cross-reactivity of the main interfering compound (genistein) was 5%, compared with 25% for the time-resolved fluoroimmunoassay.