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 Citations (Scopus)

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)681-689
Number of pages9
JournalScandinavian Journal of Clinical and Laboratory Investigation
Volume65
Issue number8
DOIs
StatePublished - Dec 2005

Fingerprint

Enzyme-Linked Immunospot Assay
Measles virus
T-cells
Viruses
Assays
T-Lymphocytes
Measles-Mumps-Rubella Vaccine
Blood
Cytokines
Data storage equipment
Blood Cells

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), 681-689. https://doi.org/10.1080/00365510500348252

Inter-operator variation in ELISPOT analysis of measles virus-specific IFN-γ-secreting T cells. / Ryan, J. E.; Ovsyannikova, I. G.; Dhiman, N.; Pinsky, N. A.; Vierkant, R. A.; Jacobson, R. M.; Poland, G. A.

In: Scandinavian Journal of Clinical and Laboratory Investigation, Vol. 65, No. 8, 12.2005, p. 681-689.

Research output: Contribution to journalArticle

Ryan, JE, Ovsyannikova, IG, Dhiman, N, Pinsky, NA, Vierkant, RA, Jacobson, RM & Poland, GA 2005, 'Inter-operator variation in ELISPOT analysis of measles virus-specific IFN-γ-secreting T cells', Scandinavian Journal of Clinical and Laboratory Investigation, vol. 65, no. 8, pp. 681-689. https://doi.org/10.1080/00365510500348252
Ryan, J. E. ; Ovsyannikova, I. G. ; Dhiman, N. ; Pinsky, N. A. ; Vierkant, R. A. ; Jacobson, R. M. ; Poland, G. A. / Inter-operator variation in ELISPOT analysis of measles virus-specific IFN-γ-secreting T cells. In: Scandinavian Journal of Clinical and Laboratory Investigation. 2005 ; Vol. 65, No. 8. pp. 681-689.
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