ELECTROPHORETIC ANALYSIS OF THE NOVEL ANTIGEN FOR THE GASTROINTESTINAL-SPECIFIC MONOCLONAL-ANTIBODY, A33

The murine monoclonal antibody A33 (mAbA33) recognises a human cell me mbrane-associated antigen selectively expressed in epithelial cells of the lower gastrointestinal tract and > 90% of colonic cancers, but is not detected in a wide range of other normal tissues by immunohistoch emical analysis. In phase I/II clinical triasl, mAbA33 has been shown to target advanced colon cancers and the humanised version is currentl y being evaluated in therapy studies. Although the mAbA33 has been wel l characterised by immunohistochemical and clinical studies, until rec ently, the target antigen has remained poorly defined. This nias large ly attributable to the antigenic determinant recognised by mAbA33 bein g dependent on the native spatial conformation of the A33 antigen whic h impeded its identification by conventional two-dimensional electroph oresis (2-DE) and immunoblot analysis. We have developed an immunoblot method, based on nonreducing/non-urea precast a-DE gels, that has fac ilitated the purification of the detergent (0.3% Triton X-100) solubil ised A33 antigen From the human colon cancer cell lines LIM1215 and SW 1222. Under these 2-DE conditions, the A33 antigen electrophoreses wit h an apparent M-r similar to 41000 and pI 5.0-6.0. Attempts to isolate the A33 antigen from 2-DE gels for direct structural analysis were un successful, due to its co-electrophoresis with actin and cytokeratin p roteins. However, using Western blot and biosensor detection the A33 a ntigen has been purified chromatographically and N-terminal sequence a nalysis was possible. Using polyclonal antibodies raised against a syn thetic peptide corresponding to the N-terminal region of the A33 antig en we have used Western blot analysis to localise the molecule in our master 2-DE protein database for normal human colon crypts and several colon carcinoma cell lines (URL address: http ://www.ludwig.edu.au). Under reducing 2-DE conditions, the A33 antigen electrophoresis as 6 d ifferentially charged isoforms (pI 4.6-4.8) with a single molecular we ight species at M-r similar to 55000.