Adapting Homogeneous Enzyme-Linked Competitive Binding Assays to Microtiter Plates

Abstract

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.