Specificity and sensitivity of commercially available assays for glucagon and oxyntomodulin measurement in humans Monika J Bak 1,2 , Nicolai Wewer Albrechtsen 1 , Jens Pedersen 1 , Bolette Hartmann 1 , Mikkel Christensen 3 , Tina Vilsbøll 3 , Filip K Knop 3 , Carolyn F Deacon 1 , Lars O Dragsted 2 and Jens J Holst 1 1 NNF Center for Basic Metabolic Research, Department of Biomedical Sciences, Faculty of Health Sciences, 2 Department of Human Nutrition, Faculty of Science, University of Copenhagen, Blegdamsvej 3, Building 12.2, DK-2200 Copenhagen, Denmark and 3 Department of Medicine, Copenhagen University Hospital Gentofte, Hellerup, DK-2900 Gentofte, Denmark Correspondence should be addressed to J J Holst Email jjholst@sund.ku.dk Abstract Aim: To determine the specificity and sensitivity of assays carried out using commercially available kits for glucagon and/or oxyntomodulin measurements. Methods: Ten different assay kits used for the measurement of either glucagon or oxyntomodulin concentrations were obtained. Solutions of synthetic glucagon (proglucagon (PG) residues 33–61), oxyntomodulin (PG residues 33–69) and glicentin (PG residues 1–69) were prepared and peptide concentrations were verified by quantitative amino acid analysis and a processing-independent in-house RIA. Peptides were added to the matrix (assay buffer) supplied with the kits (concentration range: 1.25–300 pmol/l) and to human plasma and recoveries were determined. Assays yielding meaningful results were analysed for precision and sensitivity by repeated analysis and ability to discriminate low concentrations. Results and conclusion: Three assays were specific for glucagon (carried out using the Millipore (Billerica, MA, USA), Bio-Rad (Sundbyberg, Sweden), and ALPCO (Salem, NH, USA) and Yanaihara Institute (Shizuoka, Japan) kits), but none was specific for oxyntomodulin. The assay carried out using the Phoenix (Burlingame, CA, USA) glucagon kit measured the concentrations of all three peptides (total glucagon) equally. Sensitivity and precision were generally poor; the assay carried out using the Millipore RIA kit performed best with a sensitivity around 10 pmol/l. Assays carried out using the BlueGene (Shanghai, China), USCN LIFE (Wuhan, China) (oxyntomodulin and glucagon), MyBioSource (San Diego, CA, USA) and Phoenix oxyntomodulin kits yielded inconsistent results. European Journal of Endocrinology (2014) 170, 529–538 Introduction Glucagon, oxyntomodulin and glicentin are products of the glucagon gene (GCG) located on chromosome 2q36,37 (1). The peptides arise from differential processing of the glucagon precursor, proglucagon (PG), a peptide consist- ing of 160 amino acids, but they all have the full glucagon amino acid sequence (2). Glucagon, corresponding to PG residues 33–61, is formed in the pancreas by the action of prohormone convertase (PC) 2 (3). Glicentin, correspond- ing to PG residues 1–69, is produced in the gut, where PG is processed by PC1/3 (4, 5) (Fig. 1). Part of glicentin may be cleaved to generate oxyntomodulin corresponding to PG residues 33–69 (6). Together, the intestinal products have been designated as ‘gut glucagon’, ‘gut glucagon-like immunoreactivity’ or ‘enteroglucagon’ (7). The remaining part of PG gives rise to a single large peptide, major PG fragment (PG residues 72–158), upon pancreatic proces- sing, and to the two glucagon-like peptides (GLP1 and GLP2) upon intestinal processing (3, 8, 9, 10, 11). European Journal of Endocrinology Clinical Study M J Bak and others Assays for glucagon and oxyntomodulin 170 :4 529–538 www.eje-online.org Ñ 2014 European Society of Endocrinology DOI: 10.1530/EJE-13-0941 Printed in Great Britain Published by Bioscientifica Ltd. Downloaded from Bioscientifica.com at 12/22/2021 05:59:50PM via free access