Ultrasensitive detection of proteins and peptides by capillary electrophoresis affinity assays

This work focused on development of novel methods for detection of ultra dilute solutions of proteins and peptides in biological matrices by affinity assay and capillary electrophoresis with laser induced fluorescence detection. Both competitive and noncompetitive formats of affinity assays were investigated.; A combinatorially selected DNA ligand (aptamer) against IgE was labeled with fluorophore and used as a selective fluorescent tag for determining IgE by noncompetitive format. Separations of samples containing aptamer and IgE were complete in less than 60 s and revealed two zones, corresponding to free and bound to IgE aptamer. Concentration of IgE could be determined with the detection limit of 46 pM and a linear dynamic range of 105. Similar separation conditions and application of thrombin aptamer allowed detection of thrombin.; A rapid capillary electrophoresis competitive immunoassay has been developed for the determination of glucagon. In the assay, fluorescein-conjugated glucagon is mixed with the sample followed by addition of anti-glucagon. Free and antibody-bound, tagged glucagon could be separated in 3 s using CE to obtain quantitative determination of glucagon with a concentration detection limit of 760 pM. The method was used to determine glucagon content of islets of Langerhans.; Capillary reversed-phase liquid chromatography was coupled on-line to competitive capillary electrophoresis immunoassay to improve concentration sensitivity of the assay and to provide a means for detecting multiple species that cross-react with antibody. After injection, samples were desorbed by gradient elution, mixed on-line with immunoassay reagents, incubated in a continuous-flow reaction capillary, and analyzed by capillary electrophoresis with flow-gated injections. Electrophoretic analysis of the reactor stream was performed every 1.5 s allowing nearly continuous monitoring of the RPLC separation. Preconcentration achieved by RPLC allowed improvement in the detection limit from 760 pM to 20 pM for glucagon and from 850 nM to 38 nM for neuropeptide Y. The technique was used to measure glucagon secretion from single islets of Langerhans and extracellular concentration of neuropeptide Y in rat brain...