Diagnostic accuracy of serum asialo-alpha1-acid glycoprotein concentration for the differential diagnosis of liver cirrhosis and hepatocellular carcinoma.

BACKGROUND: Serum asialoglycoproteins concentration are increased in patients with hepatic disease. We developed an antibody-lectin sandwich assay that is sensitive and specific to measure asialo-alpha(1)-acid glycoprotein (AsAGP) concentration in human serum and evaluated it as a biochemical marker for hepatic disease. METHODS: Serum AsAGP concentration was measured by antibody-lectin sandwich assay with 610 serum specimens of patients with hepatic disease. Serum from 41 healthy donors and 155 patients with non-hepatic disease served as negative controls. The AsAGP values were analyzed by receiver operator characteristics (ROC) curve analysis. The diagnostic accuracy of AsAGP value was compared with those of the conventional biochemical markers in the liver function test. RESULTS: Serum AsAGP concentration in 83% of patients with liver cirrhosis (LC) and 89% of patients with hepatocellular carcinoma (HCC) was increased over the cutoff value (1.33 microg/ml), indicating that an increase of serum AsAGP concentration is restricted to LC or HCC cases. The area under curve (AUC) in the ROC curve was 0.919 for LC and 0.946 for HCC. CONCLUSIONS: Serum AsAGP concentration exhibited good diagnostic accuracy as a biochemical marker for LC and HCC. The addition of AsAGP to conventional liver function tests may significantly improve the diagnosis and prognosis.

Development of a rapid, immunochromatographic strip test for serum asialo alpha1-acid glycoprotein in patients with hepatic disease.

Serum asialoglycoprotein (desialylated glycoproteins) concentrations have been reported to be elevated in patients with hepatic disease as compared with that of normal subjects. We recently developed a solid-phase sandwich assay for asialo alpha1-acid glycoprotein (AsAGP) as a representative of the serum asialoglycoproteins and evaluated the utility of this AsAGP as a diagnostic marker for liver cirrhosis (LC) and/or hepatocellular carcinoma (HCC). In this study, we developed a rapid, one-step immunochromatographic strip capable of specifically detecting AsAGP in serum specimens. We have produced a monoclonal antibody (mAb) to AGP, and based on ELISA and Western blot analysis, we have selected four hybridoma clones which generated mAbs to recognize AsAGP. In the immunochromatographic strip test, one mAb was used for conjugation with colloidal gold microparticles. Ricinus communis agglutinin (RCA) was immobilized onto a nitrocellulose membrane strip to form a result line in the path of chromatographic migration. Likewise, a control line was created above the result line by the immobilization of anti-mouse IgG. A serum specimen was then applied to the sample pad. The AsAGP in the sample specifically bound to the microparticles via mAb (As16.89) and co-migrated upward until the AsAGP was sandwiched with the immobilized lectin (RCA), revealing a visible result line. The colloidal gold microparticles without bound AsAGP continued to migrate, forming a visible control line. Thus, an AsAGP-positive specimen (>1.5 microg/mL) yielded a result line and a control line, whereas an AsAGP-negative specimen (<1.5 microg/mL) produced only a single control line. The entire test procedure was completed in less than 5 min. In order to examine the reliability of the testing procedures, we carried out the immunochromatographic strip test with 102 serum samples and compared the results of these tests with those obtained by ELISA. The two methods showed excellent correlation, with 83-100% above/below the cut-off value (1.5 microg/mL). Therefore, we concluded that the results of the immunochromatographic test are in excellent accordance with those of the sandwich ELISA.

Elevation of serum asialo-alpha(1) acid glycoprotein concentration in patients with hepatic cirrhosis and hepatocellular carcinoma as measured by antibody-lectin sandwich assay.

Serum asialoglycoproteins (desialylated glycoproteins) concentration was reported to be elevated in patients with hepatic disease as compared with that of normal subjects. In this study, we measured serum asialo-alpha(1) acid glycoprotein (AsAGP) level by a solid-phase sandwich assay in which monoclonal antibody (mAb) to alpha(1)-acid glycoprotein and galactose-binding lectin, ricinus communis (RCA), have been employed as capture protein and probe protein, respectively. The mAb-RCA sandwich assay was sensitive (0.02 µg/ml) and specific for AsAGP. We have determined AsAGP concentration of 869 serum specimens and analyzed the results using l.38 and 2.24 µg/ml (AsAGP) as cut-off values, respectively. AsAGP level was 0.80+/-0.29 µg/ml (mean+/-S.D.) with 97 normal serum specimens and elevated primarily in patients with liver cirrhosis (LC) or hepatocellular carcinoma (HCC). Using 1.38 µg/ml as a cutoff, 4/97 normal subjects, 11/39 acute hepatitis and 26/159 non-hepatic disease exhibited a slight elevation, whereas, AsAGP level was significantly elevated in 182/230 LC and 63/72 HCC. Meanwhile, a cutoff of 2.24 µg/ml allowed significant differentiation of LC or HCC from chronic hepatitis. Serum AsAGP level appeared to increase progressively with increasing severity of liver disease in cirrhotic patients. Thus, serum AsAGP concentration, as measured by the new mAb-RCA sandwich assay, may be a useful differential marker as a diagnostic aid for LC or HCC.

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.

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 microg/mL (AGR-GnT-III) as a cut-off value. The AGR-GnT-III level of 61 normal serum samples was 0.57 +/- 0.71 microg/ml (mean +/- SD). The results revealed an elevation in serum AGR-GnT-III levels in 60 of 86 patients (3.03 +/- 2.04 microg/ml) with liver cirrhosis (LC) and 86 of 91 patients (2.73 +/- 0.59 microg/ml) with chronic hepatitis (CH). By contrast, 3 of 61 normal subjects, 9 of 34 patients (1.02 +/- 1.03 microg/ml) with acute hepatitis and 8 of 38 patients (1.79 +/- 0.56 microg/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.