Recently devised homogeneous enzyme-linked binding assays useful for the rapid detection of carbohydrate structure/content of intact glycoproteins (via use of lectins as binders) and for quantitating given vitamins (e.g., biotin; using soluble binding proteins) are adapted successfully to a microtiter plate reader format. The problem of nonspecific adsorption of the binders and enzyme-saccharide/vitamin conjugates is solved via the addition of Tween 20 to the assay buffer. More convenient and reliable photometric detection of the preferred labeling enzyme, glucose-6-phosphate dehydrogenase (G6PDH), is accomplished by monitoring the rate of generation of reduced thio-NAD (from thio-NAD) at 405 nm instead of NADH (from NAD) at 340 nm. By employing these modifications it is shown that homogeneous enzyme-linked binding assays can be readily adapted to microtiter plates without loss in analytical assay performance. Results further suggest that other homogeneous assays based on G6PDH, including commercial EMIT assays used routinely in clinical chemistry laboratories for detecting drugs of abuse, could, in principle, be run on microtiter plates to significantly enhance sample throughput.
The use of two enzyme labels in a dual analyte simultaneous heterogeneous enzyme-linked competitive binding assay is examined. Reaction conditions for monitoring glucosed-phosphate dehydrogenase (G6PDH) and Pgalactosidase (@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,, 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 G6PDHIpgal enzyme pair for devising other dual analyte competitive and non-competitive binding assays is discussed.
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