Amplified electrochemical immunoassay for thyrotropin using thermophilic beta-NADH oxidase

The use of the highly stable, pH insensitive flavoenzyme, reduced nicotinamide adenine dinucleotide oxidase (NADH oxidase) from the thermophilic organism Thermus aquaticus in combination with alcohol dehydrogenase in an amperometric amplified immunoassay for thyrotropin (TSH) is described. NADH oxidase catalyses the oxidation of reduced nicotinamide adenine dinucleotide (NADH) with concomitant two electron reduction of di-oxygen to hydrogen peroxide. Hydrogen peroxide can be detected by oxidation at a platinum electrode poised at +650mV vs. Ag/AgCl. The enzyme amplification system described has advantages over existing amplification techniques in terms of sensitivity, specificity and operational pH dependence. The electrochemical enzyme-amplified assay for TSH was compared with a spectrophotometric enzyme-amplified system and with a non-amplified electrochemical immunoenzymometric TSH assay. The dynamic range of the electrochemical enzyme-amplified TSH immunoassay was 0.2-100 mIU/l, which was four times that of the enzyme-amplified spectrophotometric assay while the detection limits of both techniques were comparable.