Microplate immunoassay technique using polyelectrolyte carriers: kinetic studies and application to detection of the herbicide atrazine Original Research Article

Electrostatic interaction between water-soluble polyelectrolytes (poly(methacrylate) polyanion and poly(N-ethyl-4-vinylpyridinium) 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 antigen, 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 the polyelectrolyte ELISA assay, immune complexes were detected in two short steps of 8 and 5 min, whereas the traditional ELISA format required 60 min incubations to saturate binding sites of the immobilized antibodies. After optimization, the polyelectrolyte ELISA was utilized for detection of the herbicide atrazine. The sensitivity of this assay was 0.03 ng/ml, with a total duration of 40 min. The coefficient of variance of the assay (n = 4) was 1.4–8.0% for atrazine concentrations in the range 0.05–1 ng/ml.