Immunoassay techniques for detection of the herbicide simazine based on use of oppositely charged water-soluble polyelectrolytes

Linear water-soluble polyelectrolytes, i.e., poly(methacrylate) polyanion and poly(N-ethyl-4-vinylpyridinium) polycation, were used as carriers for the reactants in immunoassay, The strength of ionic forces through distance and the cooperative binding of oppositely charged chains, the carriers interact with each other at an extremely high rate and affinity. These properties of the polyelectrolytes made if possible to carry out the immunochemical steps of the assay in true solution and then to quickly separate the resulting products from the reaction mixtures, The above approach was applied to an assay for the herbicide simazine, Both enzyme-linked immunosorbent assay (ELISA) and dot blot formats of the immunoassay were evaluated. In the ELISA format, the polycation was adsorbed on the surface of a microtiter plate. A tracer antigen (simazine) was allowed to interact in solution with components of the reaction mixture containing simazine-peroxidase conjugate, specific antibodies, and staphylococcal protein A conjugated with the polyanion, and then the mixture was added to the immobilized polycation. Quick separation of the immunoreactants was achieved due to formation of interpolyelectrolyte complexes between polycation and polyanion molecules. After washing, the microplate wells were filled with a solution of substrate, and the optical density of the reaction products was measured, In the second format, a solution of the same reaction mixture (after incubation) was filtered through a porous membrane, with the polycation adsorbed. The subsequent addition of substrate led to the development of colored spots, Sensitivity of the dot blot format was close to that of the traditional ELISA format using the same reactants, i.e., 0.5 ng/mL, However, the assay was much faster (assay time decreased from 100-120 to 45 min). Sensitivities of the dot immunoassay were 1 ng/ml for densitometric detection and 10 ng/mL for visual detection with a duration of 20 min, The techniques developed here were used for simazine determination in water, milk, and juices.