Lectin-based homogeneous enzyme-linked binding assay for estimating the type and relative amount of carbohydrate...

The feasibility of using a new lectin-based homogeneous enzyme-linked binding assay for estimating the type and relative amount of specific carbohydrate structures within intact glycoproteins is examined. Malate dehydrogenase-galactose, -mannose, and -N-acetylglucosamine conjugates are utilized in conjunction with Jacalin, concanavalin A, and wheat germ agglutinin, respectively. The catalytic activity of the glyco-enzyme conjugates is inhibited significantly (greater than 60%) in solution in the presence of the respective lectins. The observed inhibition for each reagent set is reversed in proportion to the type and relative amount of specific carbohydrates present within test glycoproteins added to the assay mixture. Competitive binding ED50 values for a number of synthetic and native model glycoproteins correlate well with the known carbohydrate content of these species. The proposed method is much faster than previous solid-phase lectin-based enzyme-linked methods used to probe carbohydrate content/structure (less than 15 min) and has the potential to be fully automated.