A novel high-sensitivity electrochemiluminescence (ECL) sandwich immunoassay for the specific quantitative measurement of plasma glucagon John H. Sloan ⁎, Robert W. Siegel, Yvelina T. Ivanova-Cox, David E. Watson, Mark A. Deeg, Robert J. Konrad Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA abstract article info Article history: Received 27 June 2012 Received in revised form 24 July 2012 Accepted 27 July 2012 Available online 4 August 2012 Keywords: Glucagon Glucose Diabetes Insulin Peptide Immunoassay 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 pro- vides a robust, sensitive, and convenient method for measuring concentrations of glucagon that is highly sensi- tive and specific. This immunoassay should help to improve our understanding of the role of glucagon in the regulation of glucose metabolism. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Introduction Glucagon is a 29-residue polypeptide hormone that is produced in the pancreas by the α-cells of the islets of Langerhans [1]. The amino acid sequence of the peptide is completely conserved in mammals. Glucagon and glucagon-like peptides are transcribed from a common proglucagon gene that is expressed in the pancreas, intestine, and brain [2]. Glucagon is involved in maintaining normal levels of glucose in the blood by stimulating the liver to convert glycogen to glucose. This conversion of glycogen to glucose in concert with the opposing action of insulin to promote storage of glucose as glycogen enables the two hormones to tightly regulate blood glucose levels. Basal amounts of glucagon are essential for the maintenance of normoglycemia, and thus a key physiological role of glucagon is to prevent hypoglycemia. It has been proposed that improper expres- sion of glucagon may contribute to the development of the hypergly- cemia in diabetes [3]. Glucagon levels increase rapidly in response to hypoglycemia. Specifically, the glucagon response is the primary essential defense mechanism utilized by the body to restore blood glucose to normal levels. A complex interplay of signals is required for the proper regu- lation of glucagon secretion. These controlling factors which include glucose, intra-islet paracrine factors such as insulin and GLP-1, and the central and autonomic nervous systems interact in a coordinated fashion to regulate glucagon secretion [4,5]. In healthy subjects, glu- cagon levels increase in response to a high-protein meal and decrease in response to a high carbohydrate meal or oral glucose [6]. The glu- cagon receptor is predominantly expressed in the liver where its acti- vation leads to increased glucose production. Glucagon receptors are also expressed at lower levels in many other tissues. Recent studies have also implicated glucagon as an important component in the pro- cess of regulation of energy metabolism [7]. A radioimmunoassay to measure glucagon was first described 50 years ago [8]. Since that time there have been many advances in im- munoassay methods; however, these have not translated to a method that specifically measures glucagon without interference from cross- reacting materials [9]. In this study, we describe a highly specific electrochemiluminescent (ECL) sandwich immunoassay with superior sensitivity and recovery that provides a rapid and convenient method Clinical Biochemistry 45 (2012) 1640–1644 Abbreviations: ECL, electrochemiluminescence; RIA, radioimmunoassay; GLP, glu- cagon like peptide; TBST, Tris buffered saline with 0.05% Tween-20; BSA, bovine serum albumin; PBS, phosphate buffered saline; WT, wild-type; MRD, minimum re- quired dilution; scFv, single chain variable fragment; LLOQ, lower limit of quantitation; CDR, complementarity-determining region; SA-PE, streptavidin-R-phycoerythrin; SOE-PCR, single overlap extension PCR; VH: Ig, heavy chain variable region; VL: Ig, light chain variable region; MSD, Meso Scale Discovery; GaM-488, Alexa fluor 488 goat anti mouse IgG. ⁎ Corresponding author at: Eli Lilly and Company, Indianapolis, IN 46285, USA. Fax: +1 317 276 5281. E-mail address: Sloan_John_Harrison@lilly.com (J.H. Sloan). 0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.07.111 Contents lists available at SciVerse ScienceDirect Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem