Isolation and characterization of 5T4, a tumour‐associated antigen

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Abstract
The monoclonal antibody (MAb) 5T4 defines a human trophoblast antigen marker with a restricted pattern of expression in normal adult tissues but this antigen is expressed on a variety of carcinomas. The purification of 5T4 antigenic molecules is described from term syncytiotrophoblast by a combination of lectin‐ and immunoaffinity chromatography and gel filtration giving up to 10,000‐fold purification with 70% yield. The antigen is carried by non‐associated glycoprotein molecules with an apparent molecular weight of 72 kDa on SDS‐PAGE and a neutral pl. Removal of N‐linked sugars by N‐glycanase reveals a core protein of 42 kDa. Treatment with enzymes that cleave O‐linked sugars does not substantially alter the molecular size. The native 5T4 molecules are very resistant to proteolysis until the N‐linked sugars are removed or the glycoprotein is denatured and reduced. Glycopeptides generated by these approaches will be suitable for amino acid sequencing.