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

Preliminary Study on a Potential Panel for Quality Assurance of ELISPOT

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

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    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.
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    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
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    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.
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    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.
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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

    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