Microplate immunoassay technique using polyelectrolyte.pdfVIP

Analytica Chimica Acta 399 (1999) 151–160 Microplate immunoassay technique using polyelectrolyte carriers: kinetic studies and application to detection of the herbicide atrazine Elena V. Yazynina a , Anatoliy V. Zherdev a, Boris B. Dzantiev a , Vladimir A. Izumrudov b , Shirley J. Gee c , Bruce D. Hammock c a Institute of Biochemistry, Russian Academy of Science, Leninsky Prospect 33, 117071 Moscow, Russia b Moscow State University, Vorob’evi gori V-234, 119899 Moscow, Russia c Department of Entomology, University of California, Davis, CA 95616, USA Received 8 February 1999; received in revised form 5 May 1999; accepted 25 May 1999 Abstract Electrostatic interaction between water-soluble polyelectrolytes (poly(methacrylate) polyanion and poly(N-ethyl-4-vinyl- pyridinium) polycation) was characterized as an approach for the acceleration of microplate enzyme immunoassays (ELISAs). The proposed assay technique includes (1) carrying out of the immune interactions in the solution that contains a tracer anti- gen, antigen–peroxidase conjugate, antibodies, and polyanion–protein A conjugate, (2) binding of the polyanion-containing complexes formed with the polycation that was immobilized in microplate wells, (3) washing of the wells, and (4) optical measurement of peroxidase activity using a substrate solution. Kinetic and concentration dependencies of the assay were studied for the herbicide simazine as a model antigen. It was shown that the quantity of the interpolymeric complexes formed increased significantly within a narrow range of polyanion/polycation ratio. Either polycation alone or polycation conjugated to soybean trypsin inhibitor could be used as immobilized reactants; both variants of the assay having the same sensitivity. With