The Thomsen-Friedenreich (TF) antigen is a precursor structure of MN blood group antigens and is also expressed by about 90% of human carcinomas. The immunodominant group of TF antigen [β-galactosyl(1–3)-α-N-acetylglactosamine] is present in cryptic form in normal RBC and is revealed by neuraminidase treatment. A murine monoclonal antibody (Mab 49H.8) developed against neuraminidase treated human RBC was reactive against a variety of human tumors. We have characterized the human tumor associated TF antigen detected by this antibody from a human transitional bladder carcinoma cell line (647V), a human colon adenocarcinoma cell line (LS174T), and a pleural effusion fluid of a breast adenocarcinoma patient (PE 89). A heterologous sandwich radio-immunoassay for TF antigen was developed using Mab 49H.8 as the catcher and 125I-peanut agglutinin as the probe. Detergent extracts of 647V and LS174T cells, media conditioned by culturing these cells, and PE 89 were shown to contain the antigen by this assay. The specificity of the antigen capture by Mab 49H.8 in this assay was demonstrated by its selective inhibition by nitrophenyl-β-d-galactoside, phenyl-β-d-galactoside, and a TF hapten. Preliminary studies on TF antigen in serum samples using this assay showed that about 53.7% of the carcinoma samples contained an antigen concentration greater than 200 units/ml whereas for 90.9% of the normal samples the antigen concentration was below 200 units/ml. These studies demonstrated that the TF antigen is shed by the tumor cells both in vitro and in vivo. The TF antigen was sensitive to treatment with alkali (0.1 m NaOH for 5 h at 37°C) and periodate (10 mm sodium periodate for 1 h at room temperature), was resistant to acidic pH (50 mm acetate buffer, pH 4.5, for 5 h at 37°C), and could be extracted with perchloric acid (0.6 m for 1 h at 4°C). The antigen was shown to be a high molecular weight glycoprotein (Mr >1,000,000) by gel filtration chromatography. The density of the antigen was estimated to be about 1.35 g/ml by cesium chloride density gradient centrifugation. The antigen could be isolated from conditioned media by a combination of affinity chromatography and gel filtration with an overall purification of about 61,432-fold and a final recovery of 53.2%. Immunoprecipitation of TF antigen using Mab 49H.8 from a detergent extract of 647V cells with cell surface glycoconjugates labeled with 3H showed it to be a glycoprotein with an approximate molecular weight of 1,000,000. These studies suggest that the human TF antigen expressed by carcinoma cells and secreted into the body fluids is a mucin-like high molecular weight glycoprotein.
This work was supported by a grant from National Cancer Institute of Canada.