Establishment and characterization of a mouse monoclonal anti-fucosylceramide antibody, PC47H.

ABSTRACT

A novel mouse monoclonal anti-fucosylceramide antibody was established by using neutral glycolipids from a human pancreas cancer tissue as the immunogen. Mice were immunized with the neutral glycolipids in the form of liposome-containing lipid A. Mouse monoclonal antibodies were screened with enzyme-linked immunosorbent assay and by thin layer chromatography-immunostaining. The latter technique showed that a mouse monoclonal antibody, designated PC47H, specifically reacts with a ceramide-monoglycoside fraction of the neutral glycolipids. The effects of various monosaccharides on the reactivity of PC47H with the neutral glycolipids were tested, and it was found that only fucose was able to inhibit the binding of PC47H to the neutral glycolipids. We also examined the direct binding activity of PC47H against galactosylceramide, glucosylceramide, fucosylceramide, and ceramide. This showed that the antigen specificity of PC47H was exclusively directed against fucosylceramide. In thin layer chromatography-immunostaining experiments with neutral glycolipids prepared from various human tissues, we observed that fucosylceramide was highly expressed in human colon and gastric cancer tissues. PC47H recognized the human adenocarcinoma cell lines of colon, stomach, pancreas, and lung but did not react with other tumor cells or with several nontumorous human cells.