Inter-operator variation in ELISPOT analysis of measles virus-specific IFN-gamma-secreting T cells.
Scand J Clin Lab Invest
; 65(8): 681-9, 2005.
Article
en En
| MEDLINE
| ID: mdl-16319042
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.
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Banco de datos:
MEDLINE
Asunto principal:
Inmunoensayo
/
Linfocitos T
/
Interferón gamma
/
Virus del Sarampión
Límite:
Adolescent
/
Child
/
Female
/
Humans
/
Male
Idioma:
En
Año:
2005
Tipo del documento:
Article