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

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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, pp<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.

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