Human adenocarcinoma cells secrete and express on their cell surfaces high molecular weight, mucin-like glycoproteins that bear epitopes that are highly restricted to carcinoma cells. Monoclonal antibody B72.3, which has been extensively characterized as to reactivity with a variety of carcinomas versus normal tissues, reacts with such a mucin-like glycoprotein. The B72.3-reactive antigen, designated TAG (tumor-associated glycoprotein) -72, was partially purified and used as immunogen to produce second generation anti-TAG-72 antibodies. One of these second generation antibodies was selected to develop a procedure to purify preparative amounts of TAG-72. TAG-72, extracted from xenografted human colon carcinoma cells, was subjected to antibody affinity chromatography, size exclusion chromatography, and ion exchange chromatography. A double determinant radioimmunoassay revealed a greater than lOOO-fold purification with a greater than 10% yield. SDS-PAGE analysis of radiolabeled purified TAG-72 revealed a polydisperse, but apparently homogenous, high molecular weight glycoprotein. Amino acid analysis of the purified TAG-72 revealed a profile highly similar to that reported for other purified mucins. The purified TAG-72 has been partially deglycosylated with trifluoromethane sulfonic acid in preparation for amino acid sequence analysis.
NIH ExPorter
NIH ExPorter