title: Enzyme-amplified immunoassays.
abstract: The sensitivity of enzyme immunoassays may be enhanced by the use of enzyme-amplification. This technique uses the enzyme label in the immunoassay to provide a trigger substance for a secondary system that can generate a large quantity of coloured product. Two examples of enzyme amplifiers are described, using either a substrate cycle with phosphorylated hexose sugars, or a redox cycle involving the coenzyme NAD(+). The redox enzyme-amplifier has a detection limit of less than one attomole for the enzyme label, alkaline phosphatase. The limited dynamic range of enzyme-amplified immunoassays may be overcome by kinetic analysis of the colour development in the enzyme-amplifier, to add at least a further order of magnitude to the range of directly measured analyte concentrations in the immunoassay. This is illustrated in an enzyme-amplified immunoassay for human thyroid stimulating hormone. Amperometric measurement of the enzyme-amplifier provides a method to extend the dynamic range still further and compares favourably with the performance of a gamma counter, a luminometer or a fluorimeter.
authors.affiliation: IQ(Bio) Ltd., Downham House, Downham's Lane, Milton Road, Cambridge CB4 1XG, England.
authors.forename: C J
authors.initials: CJ
authors.lastname: Stanley
authors.forename: D H
authors.initials: DH
authors.lastname: Ellis
authors.forename: D L
authors.initials: DL
authors.lastname: Bates
authors.forename: A
authors.initials: A
authors.lastname: Johannsson