NEW APPROACH FOR SENSITIZATION OF SOLID-PHASE IMMUNOASSAY BASED ON CONTROLLING OF NONSPECIFIC-BINDING - COMBINATION OF ENZYME AMPLIFICATIONAND SENSITIVE ELECTROCHEMICAL DETECTION

An effective combination of enzymatic amplification with sensitive det ection will improve the sensitivity of solid-phase immunoassay. A seri es of approaches toward inactivating nonspecific binding sites was dev eloped to minimize the surface active sites of solid-phase matrices. F or minimizing the surface active sites, which are responsible for nons pecific binding, surfaces were coated with a lipid layer and a protein membrane, and the effects of these blocking agents were evaluated. Th ese membranes were prepared by the Langmuir-Blodgett method, and then compared with the membranes prepared by physical adsorption. Among the membranes investigated, a thin membrane of skimmed milk was best-suit ed for decreasing undesired adsorption of enzyme-labeled antibodies. H ere, alkaline phosphatase was exploited as an enzyme amplifier whose p roduct, p-nitrophenol, was easily determined either spectrophotometric ally or electrochemically. The detection limit for mouse IgG was 10(-1 1) g/ml in the case of spectrophotometric detection; in the case of el ectrochemical method, mouse IgG as low as 10(-12) g/ml was determined when nitrophenol was detected electrochemically.