[Detection of IFN-gamma level in single CD8+ T cell].

Aim: 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).

Conclusion: 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.