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

Abstract

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 (>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 ED 50 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 enzymelinked methods used to probe carbohydrate content/structure (<15 min) and has the potential to be fully automated.