Generation of polyclonal rabbit antisera to mouse melanoma associated antigens using gene gun immunization

Deborah R. Surman, Kari R. Irvine, Eliza P. Shulman, Tanir M. Allweis, Steven A. Rosenberg, Nicholas P. Restifo

Research output: Contribution to journalArticle

Abstract

Lymphocytes from patients with melanoma have been used to clone melanoma associated antigens which are, for the most part, nonmutated melanocyte tissue differentiation antigens. To establish a mouse model for the use of these 'self' antigens as targets for anti-tumor immune responses, we have employed the mouse homologues of the human melanoma antigens Tyrosinase, Tyrosinase Related Protein-1 (TRP-1), gp100, and MART-1. We sought to generate antisera against these proteins for use in the construction of experimental recombinant and synthetic anti-cancer vaccines, and for use in biologic studies. Using genes cloned from the B16 mouse melanoma or from murine melanocytes, we immunized rabbits with plasmid DNAs coated onto microscopic gold beads that were then delivered using a hand-held, helium- driven 'gene gun'. This strategy enabled us to generate polyclonal rabbit sera containing antibodies that specifically recognized each antigen, as measured by immunostaining of vaccinia virus infected cells. The sera that we generated specifically for TRP-1, gp100, and MART-1 recognized extracts of the spontaneous murine melanoma, B16. The identities of the recognized proteins was confirmed by Western blot analysis. The titers and specificities of these antisera were determined using ELISA. Interestingly, serum samples generated against murine MART-1 and gp100 developed antibodies that were cross-reactive with the corresponding human homologues. Recognition of human gp100 and murine Tyrosinase appeared to be dependent upon conformational epitopes since specificity was lost upon denaturation of the antigens. These antisera may be useful in the detection, purification and characterization of the mouse homologues of recently cloned human tumor associated antigens and may enable the establishment of an animal model of the immune consequences of vaccination against 'self' antigens.

Original languageEnglish (US)
Pages (from-to)51-62
Number of pages12
JournalJournal of Immunological Methods
Volume214
Issue number1-2
DOIs
StatePublished - May 1 1998

Keywords

  • Gene gun immunization
  • Melanoma antigen
  • Polyclonal antisera
  • Recombinant anti-cancer vaccines

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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    Surman, D. R., Irvine, K. R., Shulman, E. P., Allweis, T. M., Rosenberg, S. A., & Restifo, N. P. (1998). Generation of polyclonal rabbit antisera to mouse melanoma associated antigens using gene gun immunization. Journal of Immunological Methods, 214(1-2), 51-62. https://doi.org/10.1016/S0022-1759(98)00036-2