ELISPOT cloning of tumor antigens recognized by cytotoxic T-lymphocytes from a cDNA expression library

Akiko Uenaka, Hidenori Hata, Sanda Win, Toshiro Ono, Hisashi Wada, Eiichi Nakayama

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

The methodology of cloning genes coding for antigens recognized by T-cells from cDNA expression libraries was improved technically by using enzyme-linked immunospot (ELISPOT) assays instead of enzyme-linked immunosorbent assays (ELISA) or bioassays to detect cytokines produced by T-cells in response to antigens. Combining large and small scale ELISPOT assays for expression cloning has the following advantages compared to conventional cDNA expression cloning: i) the number of recombinant plasmids which can be screened is greater than 10,000 per well in a 24-well plate in a large scale ELISPOT assay compared to fewer than 100 per well in a 96-well plate in an IFN-gamma ELISA or a TNF-alpha bioassay; ii) the total number of recombinant plasmids which can be screened in a routine assay is 2 × 105 in only one 24-well plate in a large scale ELISPOT assay compared to 1 × 105 in ten 96-well plates in an IFN-gamma ELISA or a TNF-alpha bioassay. Thus the screening efficiency of large scale ELISPOT cloning is approximately 200 times that of conventional expression cloning approaches. The efficiency of the method was confirmed by detecting the model gene RLakt from a cDNA library of a murine leukemia RL male 1.

Original languageEnglish
Pages (from-to)1-10
Number of pages10
JournalCancer Immunity
Volume1
Publication statusPublished - Jul 13 2001

Keywords

  • ELISPOT
  • Expression cloning
  • Tumor antigens
  • cDNA library

ASJC Scopus subject areas

  • Immunology
  • Cancer Research

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    Uenaka, A., Hata, H., Win, S., Ono, T., Wada, H., & Nakayama, E. (2001). ELISPOT cloning of tumor antigens recognized by cytotoxic T-lymphocytes from a cDNA expression library. Cancer Immunity, 1, 1-10.