title: Inter-operator variation in ELISPOT analysis of measles virus-specific IFN-gamma-secreting T cells.
abstract: The ELISPOT assay is a highly sensitive technique used for the detection of individual cytokine releasing cells. We have developed an IFN-gamma ELISPOT assay utilizing unfractionated frozen peripheral blood mononuclear cells (PBMC) to quantify the frequency of measles virus (MV)-specific IFN-gamma-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-gamma-secreting T cells for each subject as determined by two different operators of an ELISPOT reader. The median frequency of MV-specific IFN-gamma-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.
authors.affiliation: Mayo Vaccine Research Group, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Ryan
authors.forename: I G
authors.initials: IG
authors.lastname: Ovsyannikova
authors.forename: N
authors.initials: N
authors.lastname: Dhiman
authors.forename: N A
authors.initials: NA
authors.lastname: Pinsky
authors.forename: R A
authors.initials: RA
authors.lastname: Vierkant
authors.forename: R M
authors.initials: RM
authors.lastname: Jacobson
authors.forename: G A
authors.initials: GA
authors.lastname: Poland
mesh_terms: D000293:Adolescent; D002648:Child; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000592:standards*; D007371:Interferon-gamma / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*; D008297:Male; D008459:Measles virus / Q000276:immunology*; D022542:Measles-Mumps-Rubella Vaccine / Q000276:immunology; D015203:Reproducibility of Results; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism*
chemical_list: D022542:Measles-Mumps-Rubella Vaccine; D007371:Interferon-gamma