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PMID: 8302824 Published · ppublish English Journal Article

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 ·Vol. 91 ·No. 3 ·1994-02-01 ·Pages 1004-8

Chen YT, Stockert E, Chen Y, Garin-Chesa P, Rettig WJ, van der Bruggen P, Boon T, Old LJ

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 anti-sera. 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.

Related Genes
MAGE-1 MAGE-2 MAGE-3
MeSH Terms
Amino Acid Sequence Animals Antibodies, Monoclonal Antibodies, Neoplasm Antigens, Neoplasm/genetics Cloning, Molecular Escherichia coli/genetics Humans Melanoma/genetics,immunology Melanoma-Specific Antigens Mice Molecular Sequence Data Neoplasm Proteins Rabbits Recombinant Proteins/genetics,immunology Sequence Homology, Amino Acid Transfection Tumor Cells, Cultured/immunology
Chemicals
Antibodies, Monoclonal Antibodies, Neoplasm Antigens, Neoplasm MAGEA1 protein, human Mageb1 protein, mouse Melanoma-Specific Antigens Neoplasm Proteins Recombinant Proteins
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Chen Y T
Ludwig Institute for Cancer Research, New York Unit, NY.
Stockert E
Chen Y
Garin-Chesa P
Rettig W J
van der Bruggen P
Boon T
Old L J
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.91.3.1004
Published
1994-02-01
Pages
1004-8
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC521442
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.91.3.1004 PMC Full Text View journal details
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