Isolation and characterization of two bladder carcinoma-associated antigens

By the use of mouse monoclonal antibodies we have earlier defined five distinct antigens associated with transitional cell carcinoma of the human urinary bladder (TCC). Two of these antigens have now been purified and partially characterized. For their purification from isolated tumor cell membranes, a rapid and efficient method of affinity chromatography was developed in which the coupling of monoclonal antibodies to protein A-Sepharose was fortified by treatment with glutaraldehyde. From these columns, 60-80% of the antigenic activity present in membrane lysates could be recovered by acid elution, corresponding to approximately 0.25-0.5 micrograms antigen protein per mg membrane protein added. The two isolated antigens are relatively hydrophobic membrane components not found in normal bladder tissue. The antigen defined by the monoclonal antibody S2C6 is confined to bladder carcinoma and B cells. It is a glycosylated, ConA-binding polypeptide with Mr 50 000 as established by SDS-PAGE. It is acidic with an IP of 3.2, heat stable up to 85 degrees C, stable at low pH (2.0) but sensitive to SDS. The antigen defined by the monoclonal antibody 7E9 is confined to bladder carcinoma but is also weakly expressed in some blood vessel endothelium. It consists of two main polypeptides of Mr 29,000 and 23,000 which do not bind to ConA. It has an IP of 7.4 and its antigenic activity is abolished by heating to 50 degrees C for 5 min. As the S2C6 antigen, it is stable at low pH but susceptible to SDS.