[Detection of IFN-gamma level in single CD8+ T cell].
OBJECTIVE
To establish a new method capillary electrophoretic immunoassay laser-induced fluorescence (CEIA-LIF), to detect IFN-gamma level in single CD8(+) T cell.


METHODS
CD8(+) T cells were isolated from peripheral blood of two patients with severe aplastic anemia(SAA) and one normal person by Ficoll-Hypaque gradient centrifugation and then were purified by immunomagnetic microbead separation. Then purified CD8(+) T cells were incubated with digitonin for 15 min followed by FITC-anti-IFN-gamma mAb for 20 min. The single cell was detected continuously by CEIA-LIF. The feasibility of the method was confirmed by inverted microscopy and laser scanning confocal fluorescence microscopy.


RESULTS
The IFN-gamma content in purified CD8(+) T cells was detected under the condition of cell-membrane integrity. The IFN-gamma level in single CD8(+) T cell from 2 SAA patients was (151.53+/-28.92)zmol and (223.72+/-45.23)zmol, respectively, and much higher that from normal control (47.47+/-17.97)zmol ( P=0.001).


CONCLUSIONS
It is feasible to quantitate IFN-gamma in single CD8(+) T cell by CEIA-LIF. CEIA-LIF might be useful in the clinical detection of intracellular cytokines.
Wang
Zhi-Jie
ZJ
Department of Hematology, Qilu Hospital,Shandong University, Jinan 250012, China. jie_969@163.com
Zhang
Hua
H
Chen
Xue-Liang
XL
Jin
Wen-Rui
WR
Cong
Ya-Qin
YQ
Han
Bo
B
Gu
Yan
Y
D000936:Antigen-Antibody Complex / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D006801:Humans; D007118:Immunoassay; D007371:Interferon-gamma / Q000032:analysis* / Q000276:immunology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D013997:Time Factors
D000936:Antigen-Antibody Complex; D007371:Interferon-gamma