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Capillary liquid chromatography of multiple peptides with on‐line capillary electrophoresis immunoassay detection
Author(s) -
German Igor,
Roper Michael G.,
Kalra Satya P.,
Rhinehart Erin,
Kennedy Robert T.
Publication year - 2001
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200109)22:17<3659::aid-elps3659>3.0.co;2-i
Subject(s) - chromatography , capillary electrophoresis , detection limit , chemistry , immunoassay , capillary action , elution , antibody , materials science , immunology , composite material , biology
A competitive immunoassay for neuropeptide Y (NPY) based on capillary electrophoresis (CE) with laser‐induced fluorescence detection was developed utilizing polyclonal antisera as the immunoreagent and fluorescein‐labeled NPY as the tracer. The assay was performed with on‐line mixing of reagents, automated injections, and a 3 s separation time. The assay had a detection limit of 850 p M . To detect NPY at lower concentrations, the assay was coupled on‐line to reversed‐phase capillary liquid chromatography (LC). In this arrangement, 5 νL samples were preconcentrated by capillary LC and eluted by a gradient of isopropanol‐containing mobile phase. The resulting chromatographic peaks were monitored by the CE immunoassay. With preconcentration, the concentration detection limit was improved to 40 p M and NPY could be measured in push‐pull perfusion samples collected from the paraventricular nucleus of freely moving rats. The technique was extended to simultaneous detection of NPY and glucagon secretion from islets of Langerhans.