Single-cell analysis by intracellular immuno-reaction and capillary electrophoresis with laser-induced fluorescence detection.
A novel method for single-cell analysis was developed by combining electroporation for intracellular immuno-reaction and capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Human interferon-gamma (IFN-gamma) in natural killer (NK) cells was chosen as the test antigen. Two forms of IFN-gamma in single cells could be well separated and detected with a limit of detection of zeptomole. In this assay, the anti-IFN-gamma monoclonal antibody labeled with fluorescein isothiocyanate (Ab*) was introduced into NK cells by electrophoration for intracellular immuno-reaction. After completion of the intracellular immuno-reaction, the NK cells were chemically pre-perforated with digitonin to lyse easily. Then, one NK cell containing the complexes of IFN-gamma isoantigens with Ab* was electrokinetically injected into the capillary. The cell adsorbed on the tip of capillary was lysed by ultrasonication. Finally, the complexes of the different forms of IFN-gamma in the cell were separated and detected by CE-LIF detection.
Zhang
Hua
H
School of Chemistry and Chemical Engineering, and State Key of Microbial Technology, Shandong University, Jinan 250100, China.
Jin
Wenrui
W
D019075:Electrophoresis, Capillary / Q000379:methods*; D018274:Electroporation; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis / Q000276:immunology; D007694:Killer Cells, Natural / Q000737:chemistry; D007834:Lasers; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D013050:Spectrometry, Fluorescence / Q000379:methods*
D011994:Recombinant Proteins; D007371:Interferon-gamma; D016650:Fluorescein-5-isothiocyanate