Cancer Immunity 1:8 (2001)

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

Akiko Uenaka¹, Hidenori Hata¹, Sanda Win¹, Toshiro Ono¹, Hisashi Wada², and Eiichi Nakayama¹

¹Department of Immunology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
²Department of Surgery, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita 565-0871, Japan

Keywords: tumor antigens, cDNA library, expression cloning, ELISPOT

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 x 10⁵ in only one 24-well plate in a large scale ELISPOT assay compared to 1 x 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.