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Sensitive determination of anti-glycolipid antibody titer and glycolipid content by an enzyme-linked immunosorbent assay (ELISA) using polystyrene beads was achieved. Glycolipid-coated polystyrene beads were used as the immobilized antigen. As antigen glycolipids, gangliotetraosylceramide (GA1), gangliotriosylceramide (GA2) and neolactotetraosylceramide (paragloboside) were used. Concentrations of 1-500 ng glycolipid in liposomes/ml or 0.1-100 micrograms glycolipid/ml could be used for the glycolipid determination. Glycolipid determination by the competitive inhibition method was not influenced by the presence of other glycolipids. A great advantage of this method is that the glycolipid-coated beads can be used repeatedly by washing the used beads with 3M NaSCN solution. The method was applied to the detection of auto-antibody against GA1 in ascitic fluid from cancer patients.

Glycolipids of human cell lines of colonic adenocarcinoma (Colo 205 and BM 314), gastric tumor (AZ 521 and KATO-III), and lung tumor (A 549) were studied by the immunohistochemical fluorescence technique, flow cytometric analysis and immunostaining on thin layer chromatoplates with antibodies against gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), fucogangliotetraosylceramide (Fuc-Gg4Cer), blood group B active lipid, globopentaosylceramide (Gb5Cer) and lactoneotetraosylceramide (nLc4Cer). Anti-nLc4Cer antibody was the only antibody which reacted with all the tumor cell lines used. The glycolipid fractions of each cell line separated by Iatrobeads column chromatography were immunostained with the six antibodies mentioned above on thin layer plates. The presence of nLc4Cer was detected in all cell lines. On the other hand, Gg4Cer was detected in gastric tumor cell lines, and Gg3Cer was detected in AZ 521. Based on these results, the tumor cell lines were analyzed by flow cytometry using anti-nLc4Cer antibody. About 70% of total cells in each cell line were separated as nLc4Cer-expressing cells. The present findings, together with the occurrence of nLc4Cer in ascitic fluids of cancer patients (Taki, T., Kojima, S., Seto, H., Yamada, H., & Matsumoto, M. (1984) J. Biochem. 96, 1257-1265), suggest that nLc4Cer may be a tumor-associated lipid.