Dual enzyme labels for simultaneous heterogeneous enzyme-linked competitive binding assays

Abstract

The use of two enzyme labels in a dual analyte simultaneous heterogeneous enzyme-linked competitive binding assay is examined. Reaction conditions for monitoring glucose-6-phosphate dehydrogenase (G6PDH) and β-galactosidase (β-gal) activities are found which enable the independent measurement of each enzyme activity in the presence of the other. The two enzymes are used as labels in the development of a model simultaneous heterogeneous competitive binding assay for detecting the biotin and vitamin B 12 content of vitamin tablets. The simultaneous assay is shown to exhibit analytical dose-response characteristics comparable to those of the single analyte assays. The potential use of the G6PDH/β-gal enzyme pair for devising other dual analyte competitive and non-competitive binding assays is discussed.