Glucagon secretion was performed by static incubation as previously described.65 (link) 15–20 size-matched isolated islets were placed into microfuge tubes and pre-incubated for 60 min at 37°C in 150 μL KRB supplemented with 3 mM glucose; pharmacological agents were added as indicated. Media was subsequently replaced with testing KRB buffer supplemented with glucose and/or pharmacological agents, as indicated. After a 60 min incubation at 37°C, the supernatant was removed and stored at −80°C before being assayed using a glucagon enzyme-linked immunosorbent assay (glucagon ELISA, 10-1271-01, Mercodia AB, Uppsala, Sweden). For content measurements, acidic ethanol was added to islets before sonication for content extraction, and storage at −20°C prior to measurement by glucagon ELISA (Mercodia).
Glucagon elisa
Manufactured by Mercodia
Sourced in Sweden, United States
The Glucagon ELISA is a quantitative analytical tool designed to measure the concentration of glucagon, a hormone produced by the pancreas that plays a key role in regulating blood glucose levels. This enzyme-linked immunosorbent assay (ELISA) provides a reliable and standardized method for the detection and quantification of glucagon in various biological samples.
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42 protocols using glucagon elisa
1
Glucagon Secretion Assay in Isolated Islets
2
Comprehensive Metabolic Profiling in Rodents
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Blood glucose was measured using a glucometer (Glutest PRO R; Sanwa Kagaku Kenkyusho Co., Ltd., Aichi, Japan). Serum non-esterified fatty acid (NEFA), triglyceride (TG), and 3-hydroxybutyrate levels were determined with NEFA C-Test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan), TG E-Test Wako (Wako Pure Chemical Industries, Ltd.), and beta Hydroxybutyrate Assay Kit (Abcam plc, Cambridge, UK), respectively. Serum alanine aminotransferase (ALT) levels were measured using Fuji Dry-chem 7000V (Fujifilm corporation, Tokyo, Japan). Urine glucose levels were analyzed with enzymatic assays in a laboratory of Oriental Yeast Co., Ltd. (Tokyo, Japan). Serum insulin and plasma glucagon levels were measured with an enzyme-linked immunosorbent assay kit (Morinaga Institute of Biological Science, Inc., Kanagawa, Japan) and Mercodia Glucagon ELISA (Mercodia AB, Uppsala, Sweden), respectively. Total lipids were extracted from the liver with chloroform and methanol (2:1 v/v), and liver TG content was assayed with TG E-Test Wako.
3
Glucagon and Incretin Hormone Measurement
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Glucagon was measured with the new ELISA, the currently used ELISA (Mercodia Glucagon ELISA, Mercodia AB, Uppsala, Sweden), and LC‐HRMS.13 In the currently used ELISA, the modified sequential protocol, with additional washing steps to improve reaction specificity16 , 17 was also used for some subjects. Glicentin and GLP‐1 were measured by use of a Glicentin ELISA kit (Mercodia) and GLP‐1 (9‐36/37) assay kit (Immuno‐Biological Laboratories, Fujioka, Japan), respectively. The other biochemical parameters were measured at the respective institutions. When the concentration of the measured sample was lower than that of the lowest concentration of the standards, it was expressed as being below the lower limit of quantification (LLOQ), and the subsequent calculation was performed using the value of the lowest concentration of the standards.
4
Metabolic Biomarker Measurement Protocol
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Glucose and triglyceride levels were measured by enzymatic assay (Iatro LQ GLU and TG II, respectively; Mitsubishi Chemical Medience, Tokyo, Japan). Insulin and C-peptide levels were measured by chemiluminescent immunoassay (Architect insulin assay and Abbott C-peptide assay; Abbott Japan Co., Ltd, Tokyo, Japan). Glucagon was measured by sandwich enzyme-linked immunosorbent assay (Mercodia glucagon ELISA; Mercodia AB, Uppsala, Sweden).
5
Insulin Aspart Pharmacokinetics and Safety
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Fat‐free mass was determined by dual‐energy X‐ray absorptiometry. Free serum IAsp concentrations (polyethylene glycol precipitated) were determined using a validated IAsp‐specific enzyme‐linked immunosorbent assay having a lower limit of quantification (LLOQ) of 10 pmol/L.
The PG concentrations were measured using a SuperGL 2 glucose analyser (Dr Müller Gerätebau GmbH, Freital, Germany) using an electrochemical method.
Plasma [6‐3H] glucose specific activity was determined using liquid scintillation counting,14 and plasma enrichment of [1‐13C] glucose and [6,6‐2H2] glucose was determined by gas chromatography‐mass spectrometry (Thermoquest),15 at the Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota.
Plasma glucagon concentrations were determined in plasma using a validated enzyme‐linked immunosorbent assay with an LLOQ of 17.7 pg/mL (Mercodia Glucagon ELISA, Mercodia AB, Uppsala, Sweden).
Safety assessments included adverse events, hypoglycaemic episodes (classified as “severe” according to the American Diabetes Association, ie, requiring third‐party assistance,16 or “confirmed”, ie, documented by PG <3.1 mmol/L, with or without symptoms consistent with hypoglycaemia), laboratory safety variables, physical examination, vital signs and ECG.
The PG concentrations were measured using a SuperGL 2 glucose analyser (Dr Müller Gerätebau GmbH, Freital, Germany) using an electrochemical method.
Plasma [6‐3H] glucose specific activity was determined using liquid scintillation counting,
Plasma glucagon concentrations were determined in plasma using a validated enzyme‐linked immunosorbent assay with an LLOQ of 17.7 pg/mL (Mercodia Glucagon ELISA, Mercodia AB, Uppsala, Sweden).
Safety assessments included adverse events, hypoglycaemic episodes (classified as “severe” according to the American Diabetes Association, ie, requiring third‐party assistance,
6
Measuring Plasma Glucagon, FGF21, Insulin, and C-peptide
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Peripheral and liver vein blood samples were immediately stored on ice until centrifugation at 4°C. After allocation in cryotubes, plasma samples were transported on dry ice and stored at −80 °C until analysis. Plasma concentrations of glucagon were analyzed using ELISA (Mercodia Glucagon ELISA, Mercodia AB) (Wewer Albrechtsen et al., 2014 (link)). FGF21 concentrations were quantified using Quantikine® Human FGF‐21 Immunoassay (Catalog Number DF2100). We measured insulin and C‐peptide concentrations by immunoassay with Cobas e 602.
7
Metabolic Phenotyping in Mouse Models
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Mice were maintained on standard chow (Teklad diet 2018) or provided a high-fat/high-sucrose (HFHS) diet (cat. no. D12451i; Research Diets) starting at 8 weeks old. Physiological tests were performed at 13–17 weeks (chow diet) or 28–30 weeks of age (HFHS diet), as indicated, or within 30 days of streptozotocin (STZ) administration. Oral glucose tolerance tests (OGTT) were performed after a 16-h fast following oral administration of glucose diluted in water (1.5 g/kg for chow-fed/HFHS and 1 g/kg for HFHS/STZ mice). Insulin tolerance tests (ITT) were performed after injection of human insulin (1 unit/kg i.p. for chow-fed and 1.5 units/kg for HFHS-fed mice; Lilly) in 4-h-fasted mice. Fasted and refed glucose and insulin were measured after 16 h fasting and 2 h chow refeeding. Blood glucose or serum insulin was measured by tail vein using a standard glucometer (FreeStyle Lite, Abbott Diabetes Care) or the mouse ultrasensitive insulin ELISA (Alpco), respectively. Glucagon levels were measured after a 16-h fast in plasma containing aprotinin by glucagon ELISA (Mercodia).
8
Fasting Blood Biomarkers Measurement
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Venous blood samples were collected from 7 to 9 am following an overnight fast of at least 8 hours. Fasting biochemical measurements described previously by our group include plasma alanine transaminase (ALT) (28 ), serum high-sensitivity C-reactive protein (hs-CRP) (29 (link)), serum insulin, serum C-peptide, plasma glucose, whole blood HbA1c (30 (link)), plasma high-density lipoprotein cholesterol (HDL-C), plasma low-density lipoprotein cholesterol (LDL-C), plasma triglycerides (31 (link)), and plasma total GLP-1 (21).
To measure glucagon, blood samples were drawn in ice-cold EDTA tubes, separated by centrifugation within 20 minutes, and stored immediately at −80 °C until further analyses. Plasma glucagon was measured using the Mercodia Glucagon ELISA (RRID: AB_2737304,https://scicrunch.org/resources/Any/search?q=AB_2737304&l=AB_2737304 , Cat. No. 10-1271-01, Uppsala, Sweden) (32 (link)). The assay was performed in duplicate and read on a SpectraMax iD3 (San Jose, CA, USA). The standard curve for glucagon had a range of 1.5 to 130 pmol/L, with a lower limit of quantification (LLOQ) of 0.75 pmol/L. Values below the LLOQ (n = 42) were assigned half the LLOQ (0.375 pmol/L). The interassay CV was 12.2% and the intra-assay CV was 12.1%.
To measure glucagon, blood samples were drawn in ice-cold EDTA tubes, separated by centrifugation within 20 minutes, and stored immediately at −80 °C until further analyses. Plasma glucagon was measured using the Mercodia Glucagon ELISA (RRID: AB_2737304,
9
Glucagon Regulation in MafA Mice
10
Metabolic Profiling of Transplanted Diabetic Mice
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