Single-cell analysis by intracellular immuno-reaction and capillary electrophoresis with laser-induced fluorescence detection

摘要

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. (c) 2005 Elsevier B.V. All rights reserved.