Journal of Microbiology and Biotechnology

  • 제18권9호
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  • Pages.1606-1612
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  • 2008
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

Detection of Expressed IL-32 in Human Stomach Cancer Using ELISA and Immunostaining

  • Seo, Eun-Hee (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kang, Jeong-Woo (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kim, Ki-Hong (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Cho, Min-Chul (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Lee, So-Jung (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kim, Hee-Jong (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kim, Jung-Hee (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kim, Eun-Jin (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Park, Dong-Ki (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kim, Soo-Hyun (Department of Biomedical Science, Konkuk University) ;
  • Choi, Yang-Kyu (College of Veterinary Medicine, Konkuk University) ;
  • Kim, Jin-Man (Department of Pathology, Chungnam National University College of Medicine) ;
  • Hong, Jin-Tae (College of Pharmacy, Chungbuk National University)
  • 발행 : 2008.09.30
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초록

Interleukin (IL)-32 is a recently identified proinflammatory cytokine that is one of the IL-18 inducible genes, and plays an important role in autoimmune and inflammatory diseases. We produced antibodies against IL-32 and studied the expression of IL-32 in human stomach cancer. We detected IL-32 secreted from K-562 cells which were stably transfected with IL-32 and in the sera of stomach cancer patients by a sandwich ELISA using a monoclonal antibody KU32-52 and a polyclonal antibody. In order to optimize a sandwich immunoassay, recombinant IL-32a was added, followed by the addition of a biotinylated KU32-52 into microtiter plate wells precoated with a goat anti-IL-32 antibody. The bound biotinylated KU32-52 was probed with a streptavidin conjugated to HRP. This sandwich ELISA was highly specific and had a minimal detection limit of 80 pg/ml (mean${\pm}$SD of zero calibrator) and measuring up to 3,000 pg/ml. This ELISA showed no cross-reaction with other cytokines such as hIL-1$\alpha$, hIL-1$\beta$, hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-$\alpha$. Intra-assay coefficients of variation were 18.5% to 4.6% (n=10), and inter-assay coefficients were 23% to 9% (n=10). The average IL-32 level in the sera of 16 stomach cancer patients (189 pg/ml) was higher than that of 12 healthy control men (109 pg/ml). Our results indicate that serum IL-32 level can be detected by using an established ELISA, and that this immunoassay and mAb KU32-09 specific for immunohistochemistry can be used in the detection of expressed and secreted IL-32 in stomach cancer patients.

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