ADAPTING HOMOGENEOUS ENZYME-LINKED COMPETITIVE-BINDING ASSAYS TO MICROTITER PLATES

Recently devised homogeneous enzyme-linked binding assays useful for t he rapid detection of carbohydrate structure/content of intact glycopr oteins (via use of lectins as binders) and for quantitating given vita mins (e.g., biotin; using soluble binding proteins) are adapted succes sfully 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 conveni ent and reliable photometric detection of the preferred labeling enzym e, glucose-6-phosphate dehydrogenase (G6PDH), is accomplished by monit oring the rate of generation of reduced thio-NAD (from thio-NAD) at 40 5 nm instead of NADH (from NAD) at 340 nm. By employing these modifica tions 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 ba sed on G6PDH, including commercial EMIT assays used routinely in clini cal chemistry laboratories for detecting drugs of abuse, could, in pri nciple, be run on microtiter plates to significantly enhance sample th roughput. (C) 1994 Academic Press, Inc.