An enzyme-linked lectin assay for α1,3-galactosyltransferase

UDP-Gal:Gal beta 1-4GlcNAc alpha 1,3-galactosyltransferase (alpha 3GalT) is responsible for the synthesis of carbohydrate xenoantigen Gal alpha 1-3Gal beta 1-4GlcNAc. In this work a convenient and sensitive assay system for quantification of alpha 3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surface had been coated with the polyacrylamide conjugate of the disaccharide Gal beta 1-4GlcNAc:(acceptor) are incubated with alpha 3GalT in the presence of cold UDP-Gal as glycosyl donor. Formation of product by enzymatic extension of the glycan chain is detected by the biotinylated plant lectin Viscum album agglutinin, The standard curve for correct quantification of alpha 3GalT activity is completed after running standard assays with no (background) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of the acceptor and the glycosyl donor UDP-Gal, In accordance with the known specificity of alpha 3GalT, no enzymatic conversion of Le(x) into Gal alpha Le(x) was observed using this assay, Human alpha Gal antibodies were isolated using a disaccharide-exposing affinity adsorbent and their specificity was studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive. (C) 2000 Academic Press.