Electrophoretic analysis of the novel antigen for the gastrointestinal-specific monoclonal antibody, A33 Journal Article
| Authors: | 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. |
| Article Title: | Electrophoretic analysis of the novel antigen for the gastrointestinal-specific monoclonal antibody, A33 |
| 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. |
| Keywords: | controlled study; human cell; sequence analysis; conference paper; protein conformation; animals; mice; cell line; tumor cells, cultured; data base; antibodies, monoclonal; amino acid sequence; molecular sequence data; membrane glycoproteins; epitope; immunoblotting; epithelium cell; cell line, transformed; tandem mass spectrometry; colon carcinoma; electrophoresis, gel, two-dimensional; molecular weight; colon; technique; oxidation-reduction; jurkat cells; 3t3 cells; polyacrylamide gel electrophoresis; epitopes; gel electrophoresis; rabbits; detergents; octoxynol; digestive system; peptide mass fingerprinting; antigen purification; humans; human; monoclonal antibody a33; human colonic proteins; two-dimensional polyacrylamide |
| Journal Title: | Electrophoresis |
| Volume: | 18 |
| Issue: | 3-4 |
| ISSN: | 0173-0835 |
| Publisher: | Wiley Blackwell |
| Date Published: | 1997-01-01 |
| Start Page: | 614 |
| End Page: | 621 |
| Language: | English |
| DOI: | 10.1002/elps.1150180345 |
| PUBMED: | 9150949 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Conference Paper -- Export Date: 17 March 2017 -- Source: Scopus |
Altmetric
Citation Impact
BMJ Impact Analytics
Related MSK Work