Simultaneous heterogeneous and homogeneous enzyme-linked competitive binding assays.

Abstract

Enzymes are now used widely as labels in many highly sensitive binding assays. Many analytes measured by these assays are determined in pairs, e. g., folate/vitamin B$\sb{12}$, TSH/T$\sb4$, etc., where quantitative data for both species is required for diagnostic purposes. These situations have sparked interest in developing assays for the simultaneous detection of multiple analytes (e. g., immunoassays, etc.). The major obstacle in developing simultaneous enzyme-linked competitive binding assays is finding enzyme labels which can be measured independently in the same solution without activation or inhibition by the second enzyme reaction. In this work, various enzyme systems were systematically investigated and pairs found which are shown to be useful for developing both simultaneous heterogeneous and homogeneous enzyme-linked binding assays. For the heterogeneous assay, glucose-6-phosphate dehydrogenase (G6PDH) from L. mesenteroides and $\beta$-galactosidase from E. coli were used as the dual enzyme labels. Reaction conditions were found that allow for their simultaneous determination by optically measuring the production rate of NADH (347 nm) and o-nitrophenol (415 nm), respectively. For the homogeneous assay, G6PDH from S. cerevisiae and malate dehydrogenase from pigeon's breast were found to be compatible by monitoring the rates of NADPH (344 nm) production and SNADH (400 nm) consumption, respectively. Assay conditions were studied to optimize the catalytic activities of both enzyme pairs. For the model simultaneous heterogeneous assay, biotin and vitamin B$\sb{12}$ were chosen as analytes, while folate and biotin were determined in the new simultaneous homogeneous assay scheme. Naturally occurring binding proteins for each vitamin (avidin (biotin), R-protein (vitamin B$\sb{12}$), and folate binding protein (folate)) were used to either bind the analyte or conjugate to a solid phase (Sepharose) in the heterogeneous assay or to modulate the catalytic activity of an enzyme-analyte conjugate in the homogeneous assay. These assay systems are shown to be useful for the rapid analysis of vitamins in multivitamin tablets; however, in principle these same enzyme pairs could also be conjugated to different analytes for use in developing simultaneous heterogeneous or homogeneous binding assays for other analyte combinations of biological or clinical importance.