Human K2 EDTA and P800 plasma (500 μl) was spiked with proglucagon 33–61, 35–61 and 36–61 stable-isotope-labeled internal standard peptides (CPC Scientific, custom order) and diluted with I buffer (25 mmol/l Tris-HCl, 25 mmol/l HEPES, 300 mmol/l NaCl, 0.1% (v/v) octyl β-D-glucopyranoside, pH 7.5).
Abstract
Background & Aim: Oxyntomodulin (Oxm) is a proglucagon-derived peptide agonist of both the GLP-1 and glucagon receptors and is a key regulator of gastric acid secretion and energy expenditure. Differential processing from proglucagon hinders assay immunoassay selectivity. Method & results: Antibody engineering was used to develop a sandwich immunoassay that selectively measures endogenous Oxm. The pre- and postprandial levels of Oxm from 19 healthy individuals over the course of 2 h were measured. Postprandial increases in Oxm occurred within minutes and levels significantly correlated with those obtained using previously published mass spectrometry assays. Conclusion: This sandwich immunoassay is appropriately sensitive and selective and is also amenable to high-throughput application for the reliable determination of endogenous levels of intact Oxm from human samples.
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