Analysis of single‐cell cultures by immunoaffinity capillary electrophoresis with laser‐induced fluorescence detection

Abstract

Neuropeptide regulation of immunological activity is becoming an important issue in both basic and clinical sciences, necessitating the need for analysis to be performed at the single-cell level. A microsampling procedure has been developed for studying secretion of biologically important peptides from neuropeptide-stimulated lymphocytes, based on microdialysis sampling coupled to immunoaffinity capillary electrophoresis (ICE), with laser-induced fluorescence (LIF) detection using a fibre-optic spectrometer and diode laser excitation. The system demonstrated a limit of detection in the high attomole (10(-18) mol/L) range with pure standards and was capable of monitoring secretion from a single cell over time. Using this system it was possible to differentiate the effects of four neuropeptides on both T and B cell release of regulatory cytokines. CD4(+) lymphocytes demonstrated a 7.5-fold increase in cytokine secretion over baseline following stimulation with substance P (SP) and calcitonin gene-related peptide (CGRP). B cells responded to CGRP and vasoactive intestinal peptide (VIP) stimulation (5.5-fold increase), but not to SP. These changes took place 12--20 h post-stimulation and, once the peak secretion had been reached, remained at that level for the duration of the experiment. This system demonstrates the ability to perform high sensitivity measurements on microsamples of biological fluids.

Capillary electrophoresis of single mammalian cells

“…In addition to the investigation of catecholamines in immunocompetent cells, Phillips et al [23] have analyzed secretion of cytokines, a diverse group of proteins and peptides, by lymphocytes using microdialysis and immunoaffinity CE. Using LIF detection, cytokines were measured from a single lymphocyte with limits of detection in the high attomole range.…”

Essential Role of IL-21 in B Cell Activation, Expansion, and Plasma Cell Generation during CD4+ T Cell-B Cell Collaboration

“…ICE analysis was performed as previously described (35,36). Fab Ab fragments were prepared from Abs specific to the cytokines of interest (R&D Systems) and immobilized onto 4 cm of the inner surfaces of a 100-mm i.d.-fused silica capillary via a disulphide linkage.…”

In‐line solid‐phase extraction preconcentration in capillary electrophoresis‐tandem mass spectrometry for the multiresidue detection of quinolones in meat by pressurized liquid extraction

“…Two types of applications have been reported using AC: characterization of metabolite and biopolymer mixtures [7][8][9][10][11] and determination of small molecules such as hypogly-cemic drugs [12], oxprenolol [13], verapamil [14], doxepin [15], terbutaline [16], ceftiofur [2], chlorophenols [17], and 3-nitrotyrosine [6]. Quantification was just attempted in case of small molecules and it was limited to only one or two compounds, using in all cases UV-Vis detection.…”