Human Hepatoma–Associated Cell Surface Antigen: Identification and Characterization by Means of Monoclonal Antibodies

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Abstract
A library of murine monoclonal antibodies reactive with human hepatoma cells was generated following immunization of Balb/c mice with an intact cloned human hepatoma cell line, designated PLC/PRF/5–NR. We report the characterization of one such IgG2a antibody, designated anti–PLC1. This antibody specifically stains parental PLC/PRF/5 cell membranes and membranes of SK–Hep 1 and Mahlavu human hepatoma cells grown in culture, using indirect immunofluorescence and horseradish immunoperoxidase techniques. A similar pattern of membranous staining was observed in solid tumors derived from the three hepatoma cell lines which were injected subcutaneously into athymic nude rats and mice. Spontaneous capping on the cell surface was observed in 7 to 30% of the three human hepatocellular carcinoma cell types when incubated in suspension with monoclonal anti–PLC1 at 37°C. Treatment of cells with trypsin or sustained growth in culture did not affect the intensity of membranous staining. Monoclonal anti–PLC1 appeared specific, and antibodies did not stain a variety of human carcinoma cell lines and primary tumors of nonhepatic origin, or several normal human and murine tissues. Purified 125I–labeled monoclonal anti–PLC1 bound specifically to the three hepatoma cell lines in culture. Specificity of the antigen–antibody reaction was demonstrated by competitive binding inhibition in experiments using unlabeled homologous antibody. Binding of 125I–anti–PLC1 was not inhibited by unlabeled monoclonal antibodies to HBsAg or to α–fetoprotein. Two hepatoma cell lines secrete a protein that specifically blocks binding of 125I–anti–PLC1 antibodies to cell surface antigenic determinants. This “hepatomaassociated” protein was subsequently purified by affinity chromatography from supernates derived from the three hepatoma cell lines. Analysis of this protein by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions revealed a major protein band at 70 to 75 kd. These findings suggest that monoclonal antibodies such as anti–PLC1 may serve as a potential reagent for serodiagnosis, histologic identification, imaging and destruction of human hepatocellular carcinoma.