CAPILLARY ELECTROPHORESIS BASED IMMUNOASSAY TO DETERMINE INSULIN CONTENT AND INSULIN-SECRETION FROM SINGLE ISLETS OF LANGERHANS

The development of a competitive immunoassay for insulin that is based on capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF) is described, The assay uses fluorescein isothiocyanate-derivatized insulin (FITC-insulin) as a tracer and the Fab fragment of an anti-insulin monoclonal antibody as the immunoreagent, Reproducibility and reliability of the assay are improved when CZE capillaries are coated with a neutral hydrophilic polymer. The assay has an average relative standard deviation of 3.4% and a detection limit of 3 nM or about 6 fg injected, Using the assay, insulin content of single islets of Langerhans was determined to be 35 +/- 4 ng, which is in good agreement with literature values. The assay was also used to determine insulin secretion from single islets, During 5-min static incubations, 3 mM glucose elicited 0.8 +/- 0.2 ng of insulin release while 16 mM glucose elicited 3.2 +/- 0.5 ng of insulin release, These values are also in good agreement with previously reported results, These applications demonstrate the capability of rapidly determining a low level of protein in a complex biological microenvironment.