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 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.
Phillips
T M
TM
Immunochemistry Laboratory, George Washington University Medical Centre, Washington, DC 20037, USA. phillipt@ors.od.nih.gov
D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007834:Lasers; D016131:Lymphocyte Subsets; D017551:Microdialysis; D013050:Spectrometry, Fluorescence / Q000379:methods*