A NEW VISUAL ENZYME-IMMUNOASSAY OF METHAMPHETAMINE USING LINEAR WATER-SOLUBLE POLYELECTROLYTES

A new visual enzyme immunoassay (EIA) technique has been developed. Op positely charged synthetic linear water-soluble polyelectrolytes (poly -N-ethyl-4-vinylpyridine as polycation and polymethacrylate as polyani on) were used as carriers for reagent immobilization. The ability of t hese molecules to form an insoluble complex was applied for the separa tion of bound and free components of the immunoassay reaction mixture. This approach was realized in methamphetamine visual EIA. In the firs t stage of the assay two specific reactions took place during incubati on of the analytical reagents with the probe to be analyzed: (1) compe tition between methamphetamine and hapten conjugated with peroxidase f or the interaction with specific antibodies and (2) interaction of the se antibodies with the protein A-polymethacrylate conjugate. As a resu lt of these reactions the (polyanion-protein A)-antibody-(hapten-perox idase) complex was formed. Then the reaction mixture was filtered thro ugh an Ultrabind membrane (0.45 mu m) with adsorbed poly-N-ethyl-4-vin ylpyridine, and the immunological complexes were immobilized to the me mbrane by electrostatic interaction. The level of peroxidase binding o n the membrane was measured by diaminobenzidene substrate. The system described was optimized to achieve both high rapidity (20 min) and an appropriate sensitivity (0.4 mu g/ml) for methamphetamine assay.