Analytical performance of luminescent immunoassays of different format forserum 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 d aidzein-specific antibodies raised in rabbits are immobilized and a daidzei n derivative is coupled to horseradish peroxidase (HRP) as a label. The HRP activity of the antibody-bound tracer is measured with an enhanced chemilu minescent system (luminol/H2O2/enhancer). The second immunoassay is based o n the use of bovine serum albumin-daidzein derivative immobilized on microt iter plates and a secondary anti-rabbit IgG-Fc fragment conjugated with 4,7 -bis(chlorosulfo-phenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Formation of the complex Eu3+-BCPDA enables time-resolved fluorescence-mod e detection of the amount of antibody bound to the immobilized antigen. Bot h methods fulfilled all the requirements of accuracy and precision. The det ection 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 differen t, because of their competitive-type mechanisms. The performance of the che miluminescence method is better, because the cross-reactivity of the main i nterfering compound (genistein) was 5%, compared with 25% for the time-reso lved fluoroimmunoassay.