Production of recombinant MART-1 proteins and specific anti-MART-1 polyclonal and monoclonal antibodies: Use in the characterization of the human melanoma antigen MART-1

Yutaka Kawakami, Jane K. Battles, Takeshi Kobayashi, Willis Ennis, Xiang Wang, Janis P. Tupesis, Francesco M. Marincola, Paul F. Robbins, Vincent J. Hearing, Matthew A. Gonda, Steven A. Rosenberg

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Abstract

Recombinant human MART-1 protein was produced by bacterial and baculoviral-insect cell expression systems. By immunization with bacterial MBP-MART-1 fusion prorein or MBP cleaved MART-1 protein, a rabbit polyclonal and two murine monoclonal antibodies specific for MART-1 were produced. These antibodies specifically detected MART-1 in immuno-precipitation, Western blotting, flow cytometric assays and in immunohistochemical analysis of tissue sections. They also stained cytoplasmic components in melanocytes and most melanoma cells in frozen or paraffin embedded tissue sections, indicating that these antibodies may be useful for the diagnosis of melanoma. One of the monoclonal antibodies M2-7 C10 recognized only human MART-1, but the other monoclonal antibody M2-9 E3 recognized both human and murine MART-1. The size of the human MART-1 molecule detected by SDS-PAGE with these antibodies was approximately 18 kDa, suggesting possible posttranslational modifications in the MART-1 protein. Subcellular fractionation studies suggested that MART-1 was present in melanosomes and endoplasmic reticulum, although known melanogenic enzymatic activities were not detected in the MART-1 protein. These reagents may be useful for biological studies on melanocytes and melanoma cells as well as for the development and monitoring of immunotherapy for patients with melanoma.

Original languageEnglish
Pages (from-to)13-25
Number of pages13
JournalJournal of Immunological Methods
Volume202
Issue number1
DOIs
Publication statusPublished - 1997 Mar 10

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Keywords

  • MART-1
  • baculovirus
  • immunotherapy
  • melanoma antigen
  • monoclonal antibody
  • recombinant protein

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

Kawakami, Y., Battles, J. K., Kobayashi, T., Ennis, W., Wang, X., Tupesis, J. P., Marincola, F. M., Robbins, P. F., Hearing, V. J., Gonda, M. A., & Rosenberg, S. A. (1997). Production of recombinant MART-1 proteins and specific anti-MART-1 polyclonal and monoclonal antibodies: Use in the characterization of the human melanoma antigen MART-1. Journal of Immunological Methods, 202(1), 13-25. https://doi.org/10.1016/S0022-1759(96)00211-6