Determination of UDP-N-acetylglucosamine:β-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:β-D-mannoside-1,4-N- acetylglucosaminyltransferase-III (GnT-III) catalyzes the addition of N-acetylglucosamine via a β-1,4-linkage to the β-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 β-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 μg/mL (AGR-GnT-III) as a cut-off value. The AGR-GnT-III level of 61 normal serum samples was 0.57 ± 0.71 μg/ml (mean ± SD). The results revealed an elevation in serum AGR-GnT-III levels in 60 of 86 patients (3.03 ± 2.04 μg/ml) with liver cirrhosis (LC) and 86 of 91 patients (2.73 ± 0.59 μg/ml) with chronic hepatitis (CH). By contrast, 3 of 61 normal subjects, 9 of 34 patients (1.02 ± 1.03 μg/ml) with acute hepatitis and 8 of 38 patients (1.79 ± 0.56 μg/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.