An enzyme-linked lectin assay for alpha 1,3-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 q uantification of alpha 3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surf ace had been coated with the polyacrylamide conjugate of the disaccharide G al beta 1-4GlcNAc:(acceptor) are incubated with alpha 3GalT in the presence of "cold" UDP-Gal as glycosyl donor. Formation of product by enzymatic ext ension of the glycan chain is detected by the biotinylated plant lectin Vis cum album agglutinin, The standard curve for correct quantification of alph a 3GalT activity is completed after running standard assays with no (backgr ound) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of t he acceptor and the glycosyl donor UDP-Gal, In accordance with the known sp ecificity of alpha 3GalT, no enzymatic conversion of Le(x) into Gal alpha L e(x) was observed using this assay, Human alpha Gal antibodies were isolate d using a disaccharide-exposing affinity adsorbent and their specificity wa s studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive. (C) 2000 Acad emic Press.