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Highly sensitive enzymatic assay method

Image Number 3 for United States Patent #4791057.

A highly sensitive quantitative assay method for a component which is 3.beta.-hydroxysteroid or 3-ketosteroid, in a specimen to be assayed, comprising causing this component in the specimen to take part in the cycling reaction ##STR1## and measuring a detectable change in the reaction system. There is thus provided a 3.beta.-hydroxysteroid - 3-ketosteroid cycling reaction using 3.beta.-hydroxysteroid oxidase, which consumes O.sub.2 and generates H.sub.2 O.sub.2 and 3-ketosteroid, with a substrate of 3.beta.-hydroxysteroid, or 3.beta.-hydroxysteroid dehydrogenase, which consumes reduced NAD(P) and generate NAD(P) and 3.beta.-hydroxysteroid, with a substrate of 3-ketosteroid. Example of specimens are specimens which contain 3-hydroxysteroid or 3-ketosteroid, or which liberate or generate such a component. By proceeding at a rate of more than ten cycles per minute and measuring the amount of a detectable change in the reaction, the component in a specimen can easily and sensitively be detected with good accuracy.

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