Lectin-based homogeneous enzyme-linked binding assays.

Abstract

The development of a simple and rapid homogeneous enzyme-linked binding assay (ELBA) method for studying lectin-carbohydrate interactions is described. The method is based on the homogeneous inhibition of catalytic activity of appropriate enzyme-saccharide conjugates by specific carbohydrate binding lectins. The saccharides are appended to malate dehydrogenase (MDH) and glucose-6-phosphate dehydrogenase (G6PDH) via the use of p-isothiocyanatophenyl derivatives of the carbohydrates. The feasibility of using such new homogeneous ELBAs for estimating the relative amount of carbohydrate structure/content of intact glycoproteins is examined. MDH-galactose, -mannose, and -N-acetylglucosamine conjugates are utilized in conjunction with Jacalin, Concanavalin A (Con A), and Wheat Germ Agglutinin (WGA), respectively. The catalytic activity of the glyco-enzyme conjugates is inhibited significantly ($>$60%) in solution in the presence of the respective lectins. The observed inhibition for each reagent set is reversed in proportion to the amount and specific carbohydrate structure present within test glycoproteins added to the assay mixture. Competitive binding ED$\sb{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 enzyme-linked methods used to probe carbohydrate structure ($<$15 min), and has the potential to be fully automated. Efforts to adapt the novel lectin-based homogeneous ELBA method to a microtiter plate detection arrangement are also reported. Appropriate choice of enzyme label (G6PDH), substrate (thio-nicotinamide dinucleotide), and reagents to reduce non-specific adsorption enables reliable kinetic assays of the labeling enzyme at 405 nm. Assays to assess carbohydrate structure/content of intact glycoproteins performed in microtiter plates correlates well to that obtained via the manual spectrophotometric procedure. Finally, the practical use of a Ricinus communis (RC) lectin/MDH-lactose conjugate system to rapidly quantitate a given glycoprotein, low density protein (LDL), is also examined. Inhibition of the MDH-lactose conjugate is reversed in an amount proportional to the concentration of LDL present. The selectivity of the assay scheme over other serum glycoproteins is assessed, as is the potential for using the reagent set for rapid estimation of LDL levels in human serum.