Electrophoretic analysis of the novel antigen for the gastrointestinal-specific monoclonal antibody, A33

Hong Ji, Robert L. Moritz, Gavin E. Reid, Gerd Ritter, Bruno Catimel, Ed Nice, Joan K. Heath, Sara J. White, Sydney Welt, Lloyd J. Old, Antony W. Burgess, Richard J. Simpson

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Abstract

The murine monoclonal antibody A33 (mAbA33) recognises a human cell membrane-associated antigen selectively expressed in epithelial cells of the lower gastrointestinal tract and >90% of colonic cancers, ut is not detected in a wide range of other normal tissues by immunohistochemical analysis. In phase I/II clinical trials, mAbA33 has been shown to target advanced colon cancers and the humanised version is currently being evaluated in therapy studies. Although the mAbA33 has been well characterised by immunohistochemical and clinical studies, until recently, the target antigen has remained poorly defined. This was largely attributable to the antigenic determinant recognised by mAbA33 being dependent on the native spatial conformation of the A33 antigen which impeded its identification by conventional two-dimensional electrophoresis (2-DE) and immunoblot analysis. We have developed an immunoblot method, based on nonreducing/non-urea precast 2-DE gels, that has facilitated the purification of the detergent (0.3% Triton X-100) solubilised A33 antigen from the human colon cancer cell lines LIM1215 and SW1222. Under these 2-DE conditions, the A33 antigen electrophoreses with an apparent M(r) ~41000 and pI 5.0-6.0. Attempts to isolate the A33 antigen from 2-DE gels for direct structural analysis were unsuccessful, due to its co-electrophoresis with actin and cytokeratin proteins. However, using Western blot and biosensor detection the A33 antigen has been purified chromatographically and N-terminal sequence analysis was possible. Using polyclonal antibodies raised against a synthetic peptide corresponding to the N-terminal region of the A33 antigen 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 differentially charged isoforms (pI 4.6-4,8) with a single molecular weight species at M(r) ~55 000.

Original languageEnglish
Pages (from-to)614-621
Number of pages8
JournalElectrophoresis
Volume18
Issue number3-4
DOIs
Publication statusPublished - 1 Mar 1997
Externally publishedYes

Keywords

  • Gel electrophoresis
  • Human colonic proteins
  • Monoclonal antibody A33
  • Peptide mass fingerprinting
  • Tandem mass spectrometry
  • Two-dimensional polyacrylamide

Cite this

Ji, H., Moritz, R. L., Reid, G. E., Ritter, G., Catimel, B., Nice, E., Heath, J. K., White, S. J., Welt, S., Old, L. J., Burgess, A. W., & Simpson, R. J. (1997). Electrophoretic analysis of the novel antigen for the gastrointestinal-specific monoclonal antibody, A33. Electrophoresis, 18(3-4), 614-621. https://doi.org/10.1002/elps.1150180345