Plasma glucagon was determined by the Vanderbilt University Mouse Metabolic Phenotyping Center (MMPC) Hormone Assay and Analytical Resources Core using an ELISA assay kit (catalog 10-1271-01, Mercodia Inc.).
Elisa assay kit
Manufactured by Mercodia
Sourced in Sweden, United States
The ELISA assay kit is a laboratory tool used for the detection and quantification of specific analytes in a sample. It utilizes enzyme-linked immunosorbent assay (ELISA) technology to measure the presence and concentration of a target substance, such as proteins, hormones, or antibodies, in a variety of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cobas e601, by Roche (1 mentions)
Daytona auto clinical analyzer, by Randox (1 mentions)
Adiponectin, by Merck Group (1 mentions)
Leptin, by Merck Group (1 mentions)
Insulin, by Mercodia (1 mentions)
Alphalisa method, by PerkinElmer (1 mentions)
Highly sensitive elisa assays, by R&D Systems (1 mentions)
8 protocols using elisa assay kit
1
Plasma Glucagon Quantification via ELISA
2
Blood Collection and Plasma Processing
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Each blood collection comprised an initial 1 mL blood draw, which was immediately discarded due to clotting, and a subsequent 10 mL blood sample, which was used for analysis. Samples were collected in appropriate K2E (EDTA) BD vacutainers (Becton, Dickinson Allschwil, Switzerland) and were processed and centrifuged according to the manufacturer's instructions to separate plasma from whole blood. Aliquots were stored in 2 ml cryovials and plasma samples for glucose analysis were immediately frozen and stored at −80°C; plasma samples for the analysis of insulin and C-peptide were stored at −20°C. After thawing the samples, assays were performed according to the manufacturer's instructions (SystemRoche/Hitachi cobas, Roche Diagnostics, Basel, Switzerland): (i) Glucose by the reference method with hexokinase Glucose HK Gen.3 (cobas c 501, Roche Diagnostics); (ii) C-peptide by ECLIA technology (cobas e 601, Roche Diagnostics). Insulin was measured by using an ELISA assay kit (Mercodia, Uppsala, Sweden) in accordance with the manufacturer's instructions. Phlebotomy and blood sample processing were carried out in accordance with institutional safety requirements for the handling of human biological specimens.
3
Quantifying Insulin and Cytokine Levels
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Insulin level was measured using an ELISA assay kit (Mercodia, Winston Salem, NC, USA). Cytokines (tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β)) were measured using ELISA kits purchased from Peprotech (Rocky Hill, NJ, USA). Tyr-phosphorylated insulin receptor substrate-1 (IRS-Tyr) was measured using an ELISA assay kit (Cell Signaling Technology, Beverly, MA, USA). Analyses were performed following the supplier’s protocols.
4
Plasma Glucose and Insulin Measurement
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Blood samples were collected and used to measure plasma glucose and insulin as described previously [11] . Briefly, samples were collected in appropriate BD vacutainers (Becton, Dickinson Allschwil, Switzerland) and were processed and centrifuged according to the manufacturer's instructions to separate plasma from whole blood. Aliquots were then stored in 2 ml cryovials and plasma samples for glucose analysis were immediately frozen and stored at -80 °C, whilst plasma samples for the analysis of insulin were stored at -20 °C. After thawing the samples, assays were performed according to the manufacturer's instructions: (i)
Glucose by the reference method with hexokinase Glucose HK Gen.3 (cobas c 501, Roche Diagnostics) and (ii) insulin by using an ELISA assay kit (Mercodia, Uppsala, Sweden). Phlebotomy and blood sample processing were carried out in accordance with institutional safety requirements for the handling of human biological specimens.
Glucose by the reference method with hexokinase Glucose HK Gen.3 (cobas c 501, Roche Diagnostics) and (ii) insulin by using an ELISA assay kit (Mercodia, Uppsala, Sweden). Phlebotomy and blood sample processing were carried out in accordance with institutional safety requirements for the handling of human biological specimens.
5
Comprehensive Metabolic Biomarker Assays
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Assays for plasma insulin, HbA1c, TG, adiponectin, C-reactive protein, alanine aminotransferase (ALT), norepinephrine, free fatty acids and glycerol were performed by Quest Diagnostics (Irving, TX or San Juan Capistrano, CA). Glucose was measured using the glucose oxidase method (YSI 2300 Glucose Analyzer; GMI, Inc). Glucagon and fibroblast growth factor (FGF) 21 were measured using ELISA assay kits (Mercodia, Winston-Salem, NC, and Abcam, Cambrige, MA, respectively).
6
Plasma Biomarker Analysis of Metabolic Health
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Blood was collected from indwelling catheters at protocol-specified time points into Ethylenediaminetetraacetic acid (EDTA) tubes and centrifuged at 12,857× g at 4 °C for 15 min to obtain plasma. Aliquots of plasma were stored immediately at −80 °C for subsequent analysis. Plasma glucose concentrations were measured using standardized enzyme-based assay kits (Cat # TR3823 and GL3815, respectively, Randox, Antrim, UK) on the Randox Daytona Auto Clinical Analyzer (Randox, Antrim, UK). Plasma insulin was assessed using an immunoturbidimetry assay (Cat# KAI071, Kamiya Biomedicals, Tukwila, WA, USA). Plasma oxidized low density lipoprotein (Ox-LDL) was measured using ELISA assay kits (Cat# 10-1143-01, Mercodia Inc., Winston Salem, NC, USA). Interleukin (IL)-6 and Monocyte chemoattractant protein (MCP)-1 in plasma samples were measured using high sensitive ELISA assay methods (Cat# HS 600B and DCP00 respectively. R&D Systems, Minneapolis, MN, USA). All assay protocols were performed according to the manufacturers’ instructions and appropriate quality controls were used as applicable. Intra and inter assay % coefficient of variation (CV) was below 10% in all the assays tested. Lipoprotein particles were analyzed for two meals (Control and Whole avocado meals) using Nuclear magnetic resonance (NMR) spectra of frozen plasma specimens by LipoScience (Raleigh, NC, USA).
7
Biomarker Analysis of Fasted Blood Samples
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Serum was frozen at −80 °C until further processing. ELISA assay kits were used to measure insulin (Mercodia, Sweden), leptin (Merck Millipore, Darmstadt, Germany) and adiponectin (Merck Millipore). Corticosterone was measured using a radioimmunoassay assay (MP Biomedicals, Santa Ana, CA, USA). Cholesterol was measured by an enzymatic-colorimetric assay (Spinreact, Girona, Spain). All procedures were performed following the recommendations of the manufacturers. The blood used to perform these measurements was collected at the time 0 of the GTT and after a period of 12 h of fasting.
8
Assessing Effects of Grape Seed Extract on Cardiometabolic Markers
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After 10-12 h of overnight fasting, fasting blood samples were collected at week 0 (baseline) and week 3, 6 and 10 of the intervention. Several analytical methods were used to assess the effects of GSE beverage consumption on plasma lipids, glucose, insulin, oxidised LDL (Ox-LDL) and soluble intercellular adhesion molecule-1 (sICAM-1). Plasma lipids and glucose concentrations were measured using standardised enzymebased assay kits (Randox) using the Randox Daytona auto clinical analyser (Randox). Plasma insulin concentrations were measured using the AlphaLISA method (PerkinElmer). Plasma Ox-LDL concentrations were measured using ELISA assay kits (Mercodia Inc.). Plasma sICAM-1 concentration was measured by highly sensitive ELISA assays (R&D Systems). All assay protocols were performed according to the manufacturers' instructions and appropriate quality controls were used as applicable. Homoeostasis model assessment for insulin resistance (HOMA-IR) was calculated using fasting insulin (μU/l) and fasting glucose (mmol/l) concentration divided by 22•5 (22) .
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