A user-friendly, highly sensitive assay to detect the IFN-gamma secretion by T cells.
OBJECTIVE
Development of a user-friendly test alternative to ELISA-based assays to detect IFN-gamma by in vitro cultured peripheral blood mononuclear cells (PBMC) stimulated with pathogen-derived antigens.


METHODS
The molecular components of an operational IFN-gamma ELISA-based test were applied in a lateral flow (LF) immuno-sandwich assay using up-converting phosphor (UCP) reporter particles. The analytical sensitivity of the UCP-LF IFN-gamma assay (ULIGA) was determined and the assay was qualitatively validated with a selection of 60 supernatants derived from PBMC cultures stimulated with M. leprae derived antigens, mitogen or medium alone.


RESULTS
ULIGA indicated an analytical sensitivity better than 2 pg/mL, and demonstrated four orders of magnitude dynamic range. The assay correlated well with the IFN-gamma ELISA.


CONCLUSIONS
ULIGA allows detection well below the cutoff value (100 pg/mL) used to define positive responses in the IFN-gamma ELISA. The test procedure is less demanding in respect to equipment and labor, and is suited for testing single samples.
Corstjens
Paul L A M
PL
Leiden University Medical Center, Department of Molecular Cell Biology, Leiden, The Netherlands. Corstjens@LUMC.NL
Zuiderwijk
Michel
M
Tanke
Hans J
HJ
van der Ploeg-van Schip
Jolien J
JJ
Ottenhoff
Tom H M
TH
Geluk
Annemiek
A
D001681:Biological Assay / Q000379:methods*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*
D007371:Interferon-gamma