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Volume 23 Issue 5
October 2008
    Citation: Chun-tao ZHANG, Yu WU, Chen-yan ZHAO, Kun-xue HONG, Chun-yu LIU, Ying WANG, Ping ZHONG, Jian-hui NIE, Xue-lin WU, You-chun WANG. Preliminary Study on a Potential Panel for Quality Assurance of ELISPOT .VIROLOGICA SINICA, 2008, 23(5) : 330-338.  http://dx.doi.org/10.1007/s12250-008-2933-2
    shu

    Preliminary Study on a Potential Panel for Quality Assurance of ELISPOT

    cstr: 32224.14.s12250-008-2933-2
    • 1.

      Department of Cell Biology, National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China

    • 2.

      Division of Virology and Immunology, National Center for AIDS Control and Prevention, Beijing 100050, China

    • 3.

      Shanghai Center for Disease Control and Prevention, Shanghai 200336, China

    • Corresponding author: You-chun WANG, wychun3@yahoo.com
    • Received Date: 15 January 2008
      Accepted Date: 29 July 2008
      Available online: 01 October 2008
    • Abstract: The ELISpot assay is increasingly used for assessing cellular immune responses in clinical trials of HIV-1 or cancer vaccines. However, to date, data from clinical trials do not consistently show that immune responses are correlated with clinical endpoints. This is due in part to the lack of assay standardization and validation across laboratories and therefore, a quality control panel is required to establish competency and comparability amongst different laboratories. In this study peripheral blood mononuclear cells (PBMCs) from healthy individuals were screened and frozen in liquid nitrogen. The recovery and viability of the PBMCs and the frequencies of interferon (IFN)-γ-secreting cells after CEF peptide pool stimulation were detected after various intervals in seven different laboratories. The recovery and viability did not differ significantly after different intervals. Although the frequencies of IFN (interferon)-γ-secreting cells among thawed PBMCs (peripheral blood mononuclear cells) fluctuated after CEF peptide pool stimulation at different intervals, they were not significantly decreased compared with those among fresh PBMCs. However, the viabilities, recoveries and frequencies of IFN-γ-secreting cells differed significantly among the seven laboratories. Our results indicate that cryopreserved PBMCs could be used as a quality control panel for ELISPOT. However, the procedures for ELISPOT need to be standardized amongst different laboratories
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      1. Arlen P, Tsang K Y, Marshall J L, et al..2000. The use of a rapid ELISPOT assay to analyze peptide-specific immune response in carcinoma patients to peptide versus recombinant poxvirus vaccines. Cancer Immunol Immunother, 49: 517-529.
          doi: 10.1007/s002620000145

      2. Asai T, Storkus W J, Whiteside T L, 2000. Evaluation of the modified ELISPOT assay for gamma interferon production in cancer patients receiving antitumor vaccines. Clin Diagn Lab Immunol, 7: 145-154.

      3. Cox J H, Ferrari G, Kalams S A, et al. 2005. Results of an ELISPOT Proficiency Panel Conducted in 11 Laboratories Participating in International Human Immuno-deficiency Virus Type 1 Vaccine Trials. AIDS R esearch and H uman R etroviruses, 21: 68-81.

      4. Currier J R, Kuta E G, Turk E, et al. 2002. A panel of MHC class Ⅰ restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays. J Immunol Methods, 260: 157-172.
          doi: 10.1016/S0022-1759(01)00535-X

      5. Doherty T M, Demissie A, Menzies D, et al. 2005. Effect of sample handling on analysis of cytokine responses to Mycobacterium tuberculosis in clinical samples using ELISA, ELISPOT, and quantitative PCR. J Immunol Methods, 298: 129-141.
          doi: 10.1016/j.jim.2005.01.013

      6. Kvarnstrom M, Jenmalm M C, Ekerfelt C. 2004. Effect of cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear cells from patients with different cytokine profiles, analysed with three common assays: an overall decrease of interleukin-4. Cryobiology, 49: 157-168
          doi: 10.1016/j.cryobiol.2004.06.003

      7. Maecker H T, Moon J, Bhatia S, et al..2005. Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT. BMC Immunol, 6:17.
          doi: 10.1186/1471-2172-6-17

      8. McCutcheon M, Wehner N, Wensky A, et al..1997. A sensitive ELISPOT assay to detect low-frequency human T lymphocytes. J Immunol Methods, 210: 149-166.
          doi: 10.1016/S0022-1759(97)00182-8

      9. Mwau M, McMichael A J, Hanke T. 2002. Design and validation of an enzyme-linked immunospot assay for use in clinical trials of candidate HIV vaccines. AIDS Res Hum Retroviruses, 18: 611-618
          doi: 10.1089/088922202760019301

      10. Power C A, Grand C L, Ismail N, et al. 1999. A valid ELISPOT assay for enumeration of ex vivo, antigenspecific, IFN-γ producing cells. J Immunol Methods, 227: 99-107
          doi: 10.1016/S0022-1759(99)00074-5

      11. Smith J G, Liu X, Kaufhold R M, et al. 2001. Development and validation of a gamma interferon ELISPOT assay for quantitation of cellular immune responses to varicella-zoster virus. Clin Diagn Lab Immunol, 8: 871-879.

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      Preliminary Study on a Potential Panel for Quality Assurance of ELISPOT

        Corresponding author: You-chun WANG, wychun3@yahoo.com
      • 1. Department of Cell Biology, National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China
      • 2. Division of Virology and Immunology, National Center for AIDS Control and Prevention, Beijing 100050, China
      • 3. Shanghai Center for Disease Control and Prevention, Shanghai 200336, China
      • Received Date: 15 January 2008
      • Accepted Date: 29 July 2008

      Abstract: Abstract: The ELISpot assay is increasingly used for assessing cellular immune responses in clinical trials of HIV-1 or cancer vaccines. However, to date, data from clinical trials do not consistently show that immune responses are correlated with clinical endpoints. This is due in part to the lack of assay standardization and validation across laboratories and therefore, a quality control panel is required to establish competency and comparability amongst different laboratories. In this study peripheral blood mononuclear cells (PBMCs) from healthy individuals were screened and frozen in liquid nitrogen. The recovery and viability of the PBMCs and the frequencies of interferon (IFN)-γ-secreting cells after CEF peptide pool stimulation were detected after various intervals in seven different laboratories. The recovery and viability did not differ significantly after different intervals. Although the frequencies of IFN (interferon)-γ-secreting cells among thawed PBMCs (peripheral blood mononuclear cells) fluctuated after CEF peptide pool stimulation at different intervals, they were not significantly decreased compared with those among fresh PBMCs. However, the viabilities, recoveries and frequencies of IFN-γ-secreting cells differed significantly among the seven laboratories. Our results indicate that cryopreserved PBMCs could be used as a quality control panel for ELISPOT. However, the procedures for ELISPOT need to be standardized amongst different laboratories

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