An enzyme-linked immunosorbent assay for N-acetylglucosaminyltransferase-V

Abstract

The development of an enzyme-linked immunosorbent assay (ELISA) for uridine 5′-diphospho- N-acetylglucosamine:αmannoside β1 → 6 N-acetylglucosaminyltransferase (GnT-V) is reported. The assay quantitates the enzymatic conversion of the specific synthetic GnT-V acceptor GlcNAcβ1 → 2Manα1 → 6Manβ-R (5) to the product GlcNAcβ1 → 2[GlcNAcβ1 → 6]Manα1 → 6Manβ-R (6) when these oligosaccharide structures were covalently attached to bovine serum albumin which was then coated on microtiter wells. Conversion of 5 to 6 was detected using a polyclonal antiserum raised against the product 6 and from which antibodies cross-reacting with acceptor 5 had been removed by affinity adsorption. GnT-V activity detected by ELISA was linearly proportional to both enzyme concentration and time under appropriate experimental conditions where 50–300 fmol of product was formed per microtiter well. GnT-V activity could be measured by ELISA in Triton X-100 extracts of hamster kidney acetone powder and in human serum. The twofold increase in GnT-V activity which is known to accompany Rous sarcoma virus transformation of baby hamster kidney cells could also be quantitated using the ELISA.