OBJECTIVES: To develop a novel, dual-monoclonal sandwich immunoassay with superior sensitivity that provides a rapid and convenient method for measuring glucagon. Glucagon is a 29-amino acid polypeptide hormone produced in the pancreas by the α-cells of the islets of Langerhans. Working in concert with insulin, glucagon is involved in regulating circulating glucose concentrations. DESIGN AND METHODS: The immunoassay utilizes Meso Scale Discovery (MSD) electrochemiluminescence (ECL) technology and two affinity-optimized monoclonal antibodies. A series of experiments was performed to determine the linear range of the assay and to evaluate sensitivity, accuracy, recovery, precision, and linearity. RESULTS: The sandwich assay was specific for glucagon and did not recognize the closely related peptide oxyntomodulin or other incretin peptides. The assay demonstrated excellent recovery, precision, and linearity, and a broad dynamic range of 0.14 pmol/L to 1950 pmol/L. In addition, assay results were highly correlated with those obtained using a previously described competitive RIA employing polyclonal antiserum. CONCLUSION: The use of affinity-optimized monoclonal antibodies in a sandwich immunoassay format provides a robust, sensitive, and convenient method for measuring concentrations of glucagon that is highly sensitive and specific. This immunoassay should help to improve our understanding of the role of glucagon in the regulation of glucose metabolism.
OBJECTIVES: To develop a novel, dual-monoclonal sandwich immunoassay with superior sensitivity that provides a rapid and convenient method for measuring glucagon. Glucagon is a 29-amino acid polypeptide hormone produced in the pancreas by the α-cells of the islets of Langerhans. Working in concert with insulin, glucagon is involved in regulating circulating glucose concentrations. DESIGN AND METHODS: The immunoassay utilizes Meso Scale Discovery (MSD) electrochemiluminescence (ECL) technology and two affinity-optimized monoclonal antibodies. A series of experiments was performed to determine the linear range of the assay and to evaluate sensitivity, accuracy, recovery, precision, and linearity. RESULTS: The sandwich assay was specific for glucagon and did not recognize the closely related peptide oxyntomodulin or other incretin peptides. The assay demonstrated excellent recovery, precision, and linearity, and a broad dynamic range of 0.14 pmol/L to 1950 pmol/L. In addition, assay results were highly correlated with those obtained using a previously described competitive RIA employing polyclonal antiserum. CONCLUSION: The use of affinity-optimized monoclonal antibodies in a sandwich immunoassay format provides a robust, sensitive, and convenient method for measuring concentrations of glucagon that is highly sensitive and specific. This immunoassay should help to improve our understanding of the role of glucagon in the regulation of glucose metabolism.
Authors: Jennifer S ten Kulve; Dick J Veltman; Liselotte van Bloemendaal; Frederik Barkhof; Carolyn F Deacon; Jens J Holst; Robert J Konrad; John H Sloan; Madeleine L Drent; Michaela Diamant; Richard G IJzerman Journal: Diabetologia Date: 2015-09-18 Impact factor: 10.122