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

Neuropeptide regulation of immunological activity is becoming an important issue in both basic and clinical sciences, necessitating the need for analy sis to be performed at the single-cell level. A microsampling procedure has been developed for studying secretion of biologically important peptides f rom neuropeptide-stimulated lymphocytes, based on microdialysis sampling co upled to immunoaffinity capillary electrophoresis (ICE), with laser-induced fluorescence (LIF) detection using a fibre-optic spectrometer and diode la ser excitation. The system demonstrated a limit of detection in the high at tomole (10(-18) mol/L) range with pure standards and was capable of monitor ing secretion from a single cell over time. Using this system it was possib le to differentiate the effects of four neuropeptides on both T and B cell release of regulatory cytokines. CB4(+) lymphocytes demonstrated a 7.5-fold increase in cytokine secretion over baseline following stimulation with su bstance P (SP) and calcitonin gene-related peptide (CGRP). B cells responde d to CGRP and vasoactive intestinal peptide (VIP) stimulation (5.5-fold inc rease), but not to SP. These changes took place 12-20 h post-stimulation an d, once the peak secretion had been reached, remained at that level for the duration of the experiment, This system demonstrates the ability to perfor m high sensitivity measurements on microsamples of biological fluids. Copyr ight (C) 2001 John Wiley & Sons, Ltd.