Characterization of Thomsen-Friedenreich Antibody Subpopulations from Normal Human Serum

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Abstract
Thomsen-Friedenreich (TF) antibodies were prepared from human serum by different enrichment procedures. This resulted in three antibody preparations all of which agglutinated neuraminidase-treated erythrocytes. On the other hand, each of the three antibody populations showed a distinct specificity pattern. Anti-TF1 antibodies could be inhibited in the hemagglutination inhibition assay by asialofetuin, asialotransferrin, asialoglyco-phorin and asialomucin. The sialylated form of these glycoproteins showed no inhibition. No significant inhibition could be achieved with several mono- or disaccharides. This suggests that anti-TF1 recognizes common structures on glycoproteins normally hidden by sialic acid. Anti-TF2 antibodies showed specificity for asialofetuin, bovine submaxillary mucin, asialomucin, asialoglycophorin, the disaccharide gal-β(l-3)N-acetyl-galactosamine (galNAc) and nitrophenyl-β-galactoside. Because asialotransferrin or unbound lactosamine were not inhibitory, we suppose that the residual common structure of the inhibitors is (gal)-galNAc-O-Ser/Thr, which is present in high amounts in submaxillary mucin. Anti-TF3 antibodies were inhibited by asialoglycophorin but not by asialomucin or asialofetuin. Strong saccharide inhibitors were gal-β(l-3)galNAc, nitrophenyl-β-galactoside, as well as galactose. Therefore, both of the antibody preparations, anti-TF2 and anti-TF3, could be inhibited by gal-β-( 1– 3)galNAc, but showed preference to one or the other sugar component of the disaccharide resulting in a differential recognition of glycoconjugate inhibitors. Anti-TF2 and anti-TF3 seem to recognize the carbohydrates in the context of a protein backbone, because gal-β-(l-3)galNAc in connection with a ceramide backbone (GMj) was not inhibitory. When tested on three human breast cancer cell lines, only anti-TF2 recognized epitopes exposed on the cell surface. We, therefore, conclude that human serum contains at least three subpopulations of TF antibodies with distinct specificities. Only anti-TF2 can detect cryptic eryth-rocyte epitopes which are also exposed on human breast cancer cell lines.