Insulin elisa

To measure serum blood markers such as insulin, triacylglycerol (TAG), and non-esterified fatty acids (NEFAs), samples were allowed to clot for 30 min at room temperature and then centrifuged at 3000 rpm for 10 min. The serum sample was dispensed into plain microtubes and stored at −80 °C until the assay was performed. For plasma glucose measurements, venous blood samples were collected in tubes containing sodium fluoride-ethylenediaminetetraacetic acid. For the measurement of plasma gastric inhibitory polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), venous blood samples were collected in tubes containing a DPP-4 inhibitor and protease inhibitor cocktail (BD, Tokyo, Japan). Thereafter, both samples were immediately centrifuged and stored at −80 °C until analysis. Enzymatic colorimetric assays were used to measure the plasma concentrations of glucose (GLU-HK (M); Shino-test Corporation, Kanagawa, Japan), serum TAG (Pure Auto S TAG-N; Sekisui Medical Company Limited, Tokyo, Japan), and serum NEFAs (Wako Pure Chemical Industries Limited). Enzyme-linked immunosorbent assays were used to measure plasma concentrations of insulin (Mercodia Insulin ELISA; Mercodia AB, Uppsala, Sweden), active GIP, and active GLP-1 (Yanaihara Institute, Inc. ELISA; Yanaihara Institute, Inc. Shizuoka, Japan).

For plasma glucose measurements, the venous blood samples were collected into tubes containing sodium fluoride-ethylenediaminetetraacetic acid. The samples were immediately centrifuged at 3000 rpm for 10 min at 4 °C and the serum was dispensed into plain microtubes and stored at −80 °C until the assay. For measurement of incretins such as GLP-1 and GIP, venous blood samples were collected in tubes containing a DPP-IV inhibitor and protease inhibitor cocktail (BD, Tokyo, Japan). To measure serum insulin, venous blood samples were collected in tubes containing clotting activators for isolation of serum. Samples were allowed to clot for 30 min at room temperature and then centrifuged and treated as described above. Enzymatic colorimetric assays were used to measure the plasma concentrations of glucose (GLU-HK (M); Shino-test Corporation, Kanagawa, Japan). Plasma concentrations of insulin (Mercodia Insulin ELISA; Mercodia AB, Uppsala, Sweden), total GIP (EMD Millipore ELISA; Merck Millipore, Merck Ltd., Danvers, MA, USA) and total GLP-1(EMD Millipore ELISA; Merck Millipore, Merck Ltd., Danvers, MA, USA) were measured by enzyme-linked immunosorbent assay.

Plasma glucose concentrations were measured enzymatically with an automated clinical chemistry analyzer (YSI 2300 Stat Plus Analyzer, YSI Incorporated, Yellow Spring, Ohio). Endogenous equine plasma insulin concentrations from the OST were determined using a commercial equine-optimized ELISA (Mercodia Equine Insulin ELISA, Mercodia AB, Uppsala, Sweden) evaluated for use in horses [19 (link)] and insulin levels were controlled with a commercial kit (Mercodia Animal Insulin Control (Low, Medium, High), Mercodia AB, Uppsala, Sweden). Plasma insulin concentrations from the continuous rate infusion of recombinant human insulin during the EHC procedures were determined using a commercial human ELISA (Mercodia Insulin ELISA, Mercodia AB, Uppsala, Sweden) and a commercial kit (Mercodia Diabetes Antigen Control (Low, High)/Human, Mercodia AB, Uppsala, Sweden) was used as a control. All analyses of plasma glucose and insulin were performed in duplicate. The mean intra-assay CVs for glucose, equine insulin (equine-optimized ELISA) and human insulin (human ELISA) were 0.4, 2.7 and 2.7 % respectively, as determined from duplicate analyses.