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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.

Objective: Glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted by intestinal L-cells which stimulates glucose-dependent insulin secretion. GLP-1 is initially secreted as the active peptide GLP-17-36/7, but rapidly undergoes cleavage by dipeptidyl peptidase 4 (DPP4) to yield the inactive form, GLP-19-36/7. Despite a reduced affinity for the GLP-1 receptor, GLP-19-36/7 may have cardioprotective properties. There is currently no described immunoassay capable of specifically measuring GLP-19-36/7., Design and Methods: We generated a monoclonal antibody specific for the N-terminal neoepitope of GLP-19-36/7. After affinity maturation, we paired this capture antibody with an anti-total GLP-1 monoclonal detection antibody to create a sandwich ELISA specific for GLP-19-36/7., Results: The sandwich ELISA was highly specific for GLP-19-36/7 and did not recognize GLP-17-36 or GLP-17-37. The ELISA exhibited a broad dynamic range and a lower limit of detection (LLOD) of 3.17ng/L. In healthy volunteers, concentrations of GLP-19-36/7 increased dramatically in the postprandial state compared to the fasted state and were markedly elevated at both 30 and 120-minute postprandial time points., Conclusions: The optimization of an N-terminal-specific monoclonal antibody for GLP-19-36/7 enabled the development of a sensitive and specific sandwich ELISA assay capable of measuring physiological concentrations of GLP-19-36/7. This ELISA may have the potential to help expand our knowledge of GLP-1 biology., (Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Glucose-dependent insulinotropic peptide (GIP) is an incretin peptide secreted by intestinal K cells that stimulates insulin secretion in a glucose-dependent manner. It is secreted as an active, intact 42-amino acid peptide GIP(1-42), which is rapidly degraded by dipeptidyl peptidase 4 to GIP(3-42), which is inactive. There is currently no described monoclonal antibody-based sandwich immunoassay to quantify concentrations of GIP(1-42), the active form of the peptide., Methods: To create a sandwich ELISA for GIP(1-42), we generated a monoclonal antibody specific for the intact N-terminus of the peptide, which was further optimized to increase its affinity. We used this antibody as a conjugate antibody in a sandwich ELISA and paired it with an anti-total GIP capture monoclonal antibody to create a dual monoclonal sandwich ELISA for GIP(1-42)., Results: The sandwich ELISA was highly specific for GIP(1-42) and did not recognize GIP(3-42). The ELISA demonstrated a broad dynamic range and a lower limit of quantification of 5 ng/L. Using the ELISA, we were able to show that GIP(1-42) concentrations in healthy volunteers increased dramatically in the postprandial state compared to the fasting state. GIP(1-42) values were correlated with total GIP values overall; however, there was substantial interindividual variation., Conclusions: The use of an N-terminal-specific monoclonal antibody in a sandwich ELISA format provides a robust and convenient method for measuring concentrations of GIP(1-42), the active form of the incretin hormone. This ELISA should help to improve our understanding of the role of GIP(1-42) in regulating glucose-dependent insulin secretion.