Enzyme amplification can enhance both the speed and the sensitivity of immunoassays

Enzyme immunoassays (EIA) are now used for the quantitation of a wide range of clinically important analytes and have, in many cases, replaced radioimmunoassays, though without improving on the sensitivity of the latter technique. We describe a general enzyme-amplification method which can be used to increase both the speed and the sensitivity of EIA. In this method, the enzyme label is used to catalyse the dephosphorylation of nicotinamide adenine dinucleotide phosphate (NADP+); the NAD+ so formed then catalytically activates an NAD+-specific redox cycle, yielding an intensely coloured formazan dye. The application of this new enzyme detection method has made possible an assay for human thyroid-stimulating hormone (TSH) with a sensitivity of 1 X 10(-5) IU/1 and a progesterone assay which takes only 15 min to complete.