Determination of UDP-N-acetylglucosamine: beta-D-mannoside-1,4-N-acetylglucosaminyltransferase-III in patients sera with chronic hepatitis and liver cirrhosis using a monoclonal antibody

Abstract

The glycoprotein UDP- N- acetylglucosamine: beta- D- mannoside- 1,4- N- acetylglucosaminyltransferase- III ( GnT- III) catalyzes the addition of N- acetylglucosamine via a beta- 1, 4- linkage to the beta- linked mannose of the trimannosyl core of N- linked glycans. It has been reported that the expression of GnT- III increases in many oncogenically transformed cells and human hepatocellular carcinoma ( HCC) tissues, and GnT- III enzyme activity in serum can be used for the detection and monitoring of primary hepatomas and hepatocellular carcinomas. A solid- phase enzyme- linked immunosorbent sandwich assay in which a polyclonal antibody ( PAb) to aglycosylrecombinant GnT- III ( AGR- GnT- III) and a monoclonal antibody ( mAb) are employed as a capture protein and probe protein, respectively, is described. The sensitivity of the PAb- mAb sandwich assay, as determined by the dose- response effect for AGR- GnT- III, was 10 ng/ ml. This assay was specific for GnT- III and did not detect beta- 1, 6- N- acetylglucosaminyltrasferase- V ( GnT- V). AGR- GnT- III concentrations in 377 serum specimens were determined by the PAb- mAb sandwich assay and the results were analyzed based on the disease category, using 1.99 mu g/ mL ( AGR- GnT- III) as a cut- off value. The AGR- GnT- III level of 61 normal serum samples was 0.57 +/- 0.71 mu g/ ml ( mean +/- SD). The results revealed an elevation in serum AGR- GnT- III levels in 60 of 86 patients ( 3.03 +/- 2.04 mu g/ ml) with liver cirrhosis ( LC) and 86 of 91 patients ( 2.73 +/- 0.59 mu g/ ml) with chronic hepatitis ( CH). By contrast, 3 of 61 normal subjects, 9 of 34 patients ( 1.02 +/- 1.03 mu g/ ml) with acute hepatitis and 8 of 38 patients ( 1.79 +/- 0.56 mu g/ ml) with a variety of non- hepatic diseases exhibited a slight increase above the cut- off value. These results indicate that serum AGR- GnT- III levels are elevated predominantly in LC or CH cases. Serum AGR- GnT- III concentration, as measured by the developed PAb- mAb sandwich assay, may be a useful differential marker as a diagnostic aid for CH and/ or LC and warrants further investigations with expanded serum panels. Published in 2003.