Abstract

The development of an enzyme-linked immunosorbent assay (ELISA) for uridine 5'-diphospho-N-acetyl-glucosamine: alpha mannoside beta 1----6 N-acetylglucosaminyltransferase (GnT-V) is reported. The assay quantitates the enzymatic conversion of the specific synthetic GnT-V acceptor GlcNAc beta 1----2Man alpha 1----6Man beta-R (5) to the product GlcNAc beta 1----2[GlcNAc-beta 1----6]Man alpha 1----6Man beta-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.