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 simultaneous quantitation of insulin, glucagon, and islet amyloid polypeptide (IAPP) secretion from islets of Langerhans. Separation buffers and conditions were optimized for resolution of fluorescein isothiocyanate (FITC)-labeled glucagon and IAPP immunoassay reagents, which were excited with the 488 nm line of an Ar⁺ laser and detected at 520 nm with a photomultiplier tube (PMT). Cy5-labeled insulin immunoassay reagents were excited by a 635 nm laser diode module and detected at 700 nm with a separate PMT. Optimum resolution was achieved with a 20 mM carbonate separation buffer at pH 9.0 using a 20 cm effective separation length with an electric field of 500 V/cm. Limits of detection for insulin, glucagon, and IAPP were 2, 3, and 3 nM, respectively. This method was used to monitor simultaneous secretion of these peptides from as few as 14 islets after incubation in 4, 11, and 20 mM glucose for 6 hours. For insulin and IAPP, a statistically significant increase in secretion levels was observed, while glucagon levels were significantly reduced in the 4 and 11 mM glucose conditions. To further demonstrate the utility of the assay, the Ca²⁺-dependent secretion of these peptides was demonstrated which agreed with published reports. The ability to examine the secretion of multiple peptides may allow for determination of regulation of secretory processes within islets of Langerhans.