AMPLIFIED ELECTROCHEMICAL IMMUNOASSAY FOR THYROTROPIN USING THERMOPHILIC BETA-NADH OXIDASE

The use of the highly stable, pH insensitive flavoenzyme, reduced nico tinamide adenine dinucleotide oxidase (NADH oxidase) from the thermoph ilic organism Thermus aquaticus in combination with alcohol dehydrogen ase in an amperometric amplified immunoassay for thyrotropin (TSH) is described. NADH oxidase catalyses the oxidation of reduced nicotinamid e adenine dinucleotide (NADH) with concomitant two electron reduction of di-oxygen to hydrogen peroxide. Hydrogen peroxide can be detected b y oxidation at a platinum electrode poised at +650mV vs. Ag/AgCl. The enzyme amplification system described has advantages over existing amp lification techniques in terms of sensitivity, specificity and operati onal pH dependence. The electrochemical enzyme-amplified assay for TSH was compared with a spectrophotometric enzyme-amplified system and wi th a non-amplified electrochemical immunoenzymometric TSH assay. The d ynamic range of the electrochemical enzyme-amplified TSH immunoassay w as 0.2-100 mIU/l, which was four times that of the enzyme-amplified sp ectrophotometric assay while the detection limits of both techniques w ere comparable.