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 a nd poly(N-ethyl-4-vinylpyridinium) polycation, were used as carriers for th e reactants in immunoassay, The strength of ionic forces through distance a nd the cooperative binding of oppositely charged chains, the carriers inter act with each other at an extremely high rate and affinity. These propertie s of the polyelectrolytes made if possible to carry out the immunochemical steps of the assay in true solution and then to quickly separate the result ing 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 ELI SA format, the polycation was adsorbed on the surface of a microtiter plate . A tracer antigen (simazine) was allowed to interact in solution with comp onents of the reaction mixture containing simazine-peroxidase conjugate, sp ecific antibodies, and staphylococcal protein A conjugated with the polyani on, and then the mixture was added to the immobilized polycation. Quick sep aration of the immunoreactants was achieved due to formation of interpolyel ectrolyte complexes between polycation and polyanion molecules. After washi ng, 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 t hrough a porous membrane, with the polycation adsorbed. The subsequent addi tion of substrate led to the development of colored spots, Sensitivity of t he 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 (as say time decreased from 100-120 to 45 min). Sensitivities of the dot immuno assay were 1 ng/ml for densitometric detection and 10 ng/mL for visual dete ction with a duration of 20 min, The techniques developed here were used fo r simazine determination in water, milk, and juices.