Identification of the Mage-1 Gene-product By Monoclonal and Polyclonal Antibodies
(1994) Proceedings of the National academy of sciences of the United States of America — Vol. 91, n° 3, p. 1004-1008 (1994)
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- Abstract
- The human MAGE-1 gene encodes a melanoma peptide antigen recognized by autologous cytotoxic T lymphocytes. To produce antibodies against the MAGE-1 gene product, several approaches were taken. Three oligopeptides were synthesized based on predicted MAGE-1 amino acid sequences and were used to generate rabbit anti-peptide antisera. In addition, a truncated MAGE-1 cDNA was cloned into an Escherichia coli expression vector, and recombinant protein was produced and purified. All three rabbit anti-peptide antisera showed reactivity against the immunizing peptide, and one reacted with the recombinant MAGE-1 protein by immunoblotting, but none reacted with cell lysates from MAGE-1 mRNA-positive cells. The recombinant MAGE-1 protein was then used for the generation of mouse monoclonal and rabbit polyclonal antibodies. One IgG1 monoclonal antibody, MA454, as well as rabbit polyclonal antisera recognized a 46-kDa protein in extracts of MAGE-1 mRNA-positive melanoma cell lines. The antibodies showed no apparent cross-reactivity with products of the closely related MAGE-2 and MAGE-3 genes. Serological typing of normal and tumor cell lysates was in full agreement with mRNA analysis, showing expression of MAGE-1 protein in MAGE-1 mRNA-positive testis and a subset of melanomas but not in MAGE-1 mRNA-negative normal or tumor tissues. Transfection of the MAGE-1 gene into a MAGE-1 mRNA-negative melanoma cell line resulted in the expression of the 46-kDa protein, confirming the identity of this protein as the MAGE-1 gene product.
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Citations
Chen, Y.-T., Stockert, E., Chen, Y., Garin-Chesa, P., Rettig, W. J., van der Bruggen, P., Boon-Falleur, T., & Old, L. J. (1994). Identification of the Mage-1 Gene-product By Monoclonal and Polyclonal Antibodies. Proceedings of the National academy of sciences of the United States of America, 91(3), 1004-1008. https://doi.org/10.1073/pnas.91.3.1004 (Original work published 1994)