Abstract
A miniaturized method for detection of antigen induced secretion of IFNg by specific T cells cultured in 384 well plates has been recently reported. In order to confidently apply this assay to clinical investigations for monitoring of specific T cell immunity, an intralaboratory validation study has been undertaken. High reproducibility and linearity of reference curves was demonstrated. Consecutive replicate experiments handled by different operators using broad panels of recall antigens were reproducible when tested on individual biological samples. Kinetics of IFNg secretion with different antigens showed a plateau after 24. h culture. Similar trends were observed with secretion of TNFa, GM-CSF and IL17, suggesting that the same kinetics can be applied if other cytokines are tested with this assay. It was demonstrated that frozen-thawed cells can be tested by cell-ELISA and that when PBMC are replaced by whole blood similar reactivity profiles were observed even though cytokine concentration was lower. T cell responses were higher in round bottom than in flat bottom wells, but these plates could not be applied to cell-ELISA as clear plates are not available for scanning. In conclusion, the assay proved flexible, since plates can be frozen at different times during the process, fresh or frozen PBMC and PBMC or whole blood could be used, and robust, since reproducibility was remarkable even when different operators performed the procedures.
| Original language | English |
|---|---|
| Pages (from-to) | 68-75 |
| Number of pages | 8 |
| Journal | Journal of Immunological Methods |
| Volume | 355 |
| Issue number | 1-2 |
| DOIs | |
| Publication status | Published - Apr 2010 |
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Keywords
- Assay validation
- Cell-ELISA
- Cytokine secretion
- Recall antigens
- T cell assay
ASJC Scopus subject areas
- Immunology
- Immunology and Allergy
Cite this
Validation of a miniaturized assay based on IFNg secretion for assessment of specific T cell immunity. / Li Pira, Giusi; Ivaldi, Federico; Moretti, Paolo; Risso, Marco; Tripodi, Gino; Manca, Fabrizio.
In: Journal of Immunological Methods, Vol. 355, No. 1-2, 04.2010, p. 68-75.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Validation of a miniaturized assay based on IFNg secretion for assessment of specific T cell immunity
AU - Li Pira, Giusi
AU - Ivaldi, Federico
AU - Moretti, Paolo
AU - Risso, Marco
AU - Tripodi, Gino
AU - Manca, Fabrizio
PY - 2010/4
Y1 - 2010/4
N2 - A miniaturized method for detection of antigen induced secretion of IFNg by specific T cells cultured in 384 well plates has been recently reported. In order to confidently apply this assay to clinical investigations for monitoring of specific T cell immunity, an intralaboratory validation study has been undertaken. High reproducibility and linearity of reference curves was demonstrated. Consecutive replicate experiments handled by different operators using broad panels of recall antigens were reproducible when tested on individual biological samples. Kinetics of IFNg secretion with different antigens showed a plateau after 24. h culture. Similar trends were observed with secretion of TNFa, GM-CSF and IL17, suggesting that the same kinetics can be applied if other cytokines are tested with this assay. It was demonstrated that frozen-thawed cells can be tested by cell-ELISA and that when PBMC are replaced by whole blood similar reactivity profiles were observed even though cytokine concentration was lower. T cell responses were higher in round bottom than in flat bottom wells, but these plates could not be applied to cell-ELISA as clear plates are not available for scanning. In conclusion, the assay proved flexible, since plates can be frozen at different times during the process, fresh or frozen PBMC and PBMC or whole blood could be used, and robust, since reproducibility was remarkable even when different operators performed the procedures.
AB - A miniaturized method for detection of antigen induced secretion of IFNg by specific T cells cultured in 384 well plates has been recently reported. In order to confidently apply this assay to clinical investigations for monitoring of specific T cell immunity, an intralaboratory validation study has been undertaken. High reproducibility and linearity of reference curves was demonstrated. Consecutive replicate experiments handled by different operators using broad panels of recall antigens were reproducible when tested on individual biological samples. Kinetics of IFNg secretion with different antigens showed a plateau after 24. h culture. Similar trends were observed with secretion of TNFa, GM-CSF and IL17, suggesting that the same kinetics can be applied if other cytokines are tested with this assay. It was demonstrated that frozen-thawed cells can be tested by cell-ELISA and that when PBMC are replaced by whole blood similar reactivity profiles were observed even though cytokine concentration was lower. T cell responses were higher in round bottom than in flat bottom wells, but these plates could not be applied to cell-ELISA as clear plates are not available for scanning. In conclusion, the assay proved flexible, since plates can be frozen at different times during the process, fresh or frozen PBMC and PBMC or whole blood could be used, and robust, since reproducibility was remarkable even when different operators performed the procedures.
KW - Assay validation
KW - Cell-ELISA
KW - Cytokine secretion
KW - Recall antigens
KW - T cell assay
UR - http://www.scopus.com/inward/record.url?scp=77951208622&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77951208622&partnerID=8YFLogxK
U2 - 10.1016/j.jim.2010.02.010
DO - 10.1016/j.jim.2010.02.010
M3 - Article
C2 - 20193687
AN - SCOPUS:77951208622
VL - 355
SP - 68
EP - 75
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 1-2
ER -