Inter-operator variation in ELISPOT analysis of measles virus-specific IFN-γ-secreting T cells

J. E. Ryan, I. G. Ovsyannikova, N. Dhiman, N. A. Pinsky, R. A. Vierkant, R. M. Jacobson, G. A. Poland

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

The ELISPOT assay is a highly sensitive technique used for the detection of individual cytokine releasing cells. We have developed an IFN-γ ELISPOT assay utilizing unfractionated frozen peripheral blood mononuclear cells (PBMC) to quantify the frequency of measles virus (MV)-specific IFN-γ-secreting T cells in 117 healthy children who had been previously immunized with two doses of the measles-mumps-rubella vaccine. We have also estimated the variability associated with the quantification of ELISPOT plates and compared the number of MV-specific IFN-γ-secreting T cells for each subject as determined by two different operators of an ELISPOT reader. The median frequency of MV-specific IFN-γ-producing memory T cells detected by this assay was 0.005 % and 0.01 % as determined by an in-house and commercial operator, respectively. Although we found a significant correlation (r=0.83, p < 0.0001) between the number of spots counted by the commercial and in-house operators of an ELISPOT reader, the median number of spots counted by the commercial operator was twice the number of spots counted by an in-house operator (p < 0.001). This demonstrates the importance of using a common ELISPOT reader and operator, among other parameters, to quantify the number of spots when a large volume of plates are being scanned and analyzed.

Original languageEnglish (US)
Pages (from-to)681-690
Number of pages10
JournalScandinavian Journal of Clinical and Laboratory Investigation
Volume65
Issue number8
DOIs
StatePublished - Dec 2005

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Keywords

  • Cytokines
  • IFN-gamma
  • Measles virus
  • Measles-mumps-rubella vaccine

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Ryan, J. E., Ovsyannikova, I. G., Dhiman, N., Pinsky, N. A., Vierkant, R. A., Jacobson, R. M., & Poland, G. A. (2005). Inter-operator variation in ELISPOT analysis of measles virus-specific IFN-γ-secreting T cells. Scandinavian Journal of Clinical and Laboratory Investigation, 65(8), 681-690. https://doi.org/10.1080/00365510500348252