Simultaneous capillary electrophoresis competitive immunoassay for insulin, glucagon, and islet amyloid polypeptide secretion from mouse islets of Langerhans

摘要

A capillary electrophoresis competitive immunoassay was developed for the simultaneous quantitation of insulin, glucagon, and islet amyloid polypeptide (IAPP) secretion from islets of Langerhans. Separation buffers and conditions were optimized for the resolution of fluorescein isothiocyanate (FITC)-labeled glucagon and IAPP immunoassay reagents, which were excited with the 488nm line of an Ar+ laser and detected at 520nm with a photomultiplier tube (PMT). Cy5-labeled insulin immunoassay reagents were excited by a 635nm laser diode module and detected at 700nm with a separate PMT. Optimum resolution was achieved with a 20mM carbonate separation buffer at pH 9.0 using a 20cm effective separation length with an electric field of 500V/cm. Limits of detection for insulin, glucagon, and IAPP were 2, 3, and 3nM, respectively. This method was used to monitor the simultaneous secretion of these peptides from as few as 14 islets after incubation in 4, 11, and 20mM glucose for 6h. For insulin and IAPP, a statistically significant increase in secretion levels was observed, while glucagon levels were significantly reduced in the 4 and 11mM glucose conditions. To further demonstrate the utility of the assay, the Ca~(2+)-dependent secretion of these peptides was demonstrated which agreed with published reports. The ability to examine the secretion of multiple peptides may allow for the determination of regulation of secretory processes within islets of Langerhans.