In FWS, glucose and insulin concentrations were measured in serum using the Roche Cobas 6000 analyzer (Roche Diagnostics, Indianapolis, IN). In the UPMC CHP cohort, plasma glucose was determined by the glucose oxidase method using a glucose analyzer (Yellow Springs Instrument Co., Yellow Springs, OH, USA), and insulin by commercially available radioimmunoassay (Millipore, St. Charles, MO, USA), as previously reported ( 15). HbA1c was measured by high-performance liquid chromatography (Tosoh Medics, Inc., San Francisco, CA, USA). In the CU study, blood glucose was measured using StatStrip ® Hospital Glucose Monitoring System (Novo Biomedical) except for AIRS (Yellow Springs Instrument Co., Yellow Springs, OH, US). Serum insulin was determined with radioimmunoassay (EMD Millipore, Billerica, MA), HbA1c was measured by highperformance liquid chromatography (Tosoh Medics, Inc., San Francisco, CA, USA) or with a handheld analyzer.
Radioimmunoassay
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
Radioimmunoassay is a sensitive analytical technique used to measure the concentration of specific substances, such as hormones, in a sample. It combines the use of radioactive isotopes and the highly specific interaction between an antigen and its corresponding antibody to quantify the target analyte. The core function of radioimmunoassay is to provide accurate and precise measurements of trace amounts of analytes in complex matrices.
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Lab products found in correlation
Dcct calibrated ion exchange high performance liquid chromatography, by Bio-Rad (4 mentions)
High performance liquid chromatography (hplc), by Tosoh (2 mentions)
Radioimmunoassay, by Linco Research (2 mentions)
Nefa c test kit, by Fujifilm (2 mentions)
Biosen c line plus glucose analyzer, by EKF Diagnostics (2 mentions)
Atellica system, by Siemens (2 mentions)
Gas chromatographic mass spectrometry, by Thermo Fisher Scientific (2 mentions)
Access assay, by Beckman Coulter (2 mentions)
Biosen apparatus, by EKF Diagnostics (2 mentions)
Au400e chemistry analyzer, by Olympus (2 mentions)
Enzymatic colorimetric assay, by Fujifilm (2 mentions)
Vitros 5.1 fs chemistry system, by Ortho Clinical Diagnostics (2 mentions)
G7 automated hplc analyzer, by Tosoh (2 mentions)
Vitros 5600, by Ortho Clinical Diagnostics (2 mentions)
Dca vantage, by Siemens (2 mentions)
Quant it picogreen, by Thermo Fisher Scientific (2 mentions)
Plasma glycerol, by R-Biopharm (2 mentions)
Glucose oxidase method, by Analox Instruments (2 mentions)
Sector imager 2400, by Mesoscale (2 mentions)
Chemiluminescent immunoassay, by DiaSorin (2 mentions)
111 protocols using radioimmunoassay
1
Multisite Glycemic Biomarker Measurement
2
Plasma Biomarker Analysis Methodology
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Plasma glucose was determined with the glucose oxidase method using a Biosen C-line plus glucose analyzer (EKF Diagnostics, Barleben/Magdeburg, Germany). Plasma insulin was determined by immunoassay on an Atellica system (Siemens, Erlangen, Germany) with intra-assay variation of 3% and inter-assay variation of 7%. Plasma FFAs were determined by an enzymatic colorimetric method (NEFA C test kit; Wako Chemicals, Neuss, Germany) with intra-assay variation of 1% and inter-assay variation of 4–15%. Plasma glucagon was determined by radioimmunoassay (Linco Research, St Charles, MO, USA) with intra-assay variation of 4–8% and inter-assay variation of 6–11%. Plasma leptin was determined by radioimmunoassay (Millipore, Burlington, MA, USA) with intra-assay and inter-assay variation of 6%. Plasma ghrelin was determined by radioimmunoassay (Millipore, Burlington, MA, USA) with intra-assay variation of 4% and inter-assay variation of 6%.
3
Biomarker Analysis in High-Sugar Diet
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Blood samples were collected before initiating the high-sugar diet, 2 days before surgical intervention, and at 3 and 6 weeks post-intervention. Animals were fasted overnight for no longer than 16 h before sampling. Measurement of all biochemical parameters was completed at the University of California Davis, Department Nutrition Assay Services Unit. Plasma insulin concentrations were quantified with a radioimmunoassay (Millipore, St. Charles, MO, USA), and plasma glucose concentrations were determined using a YSI Glucose Analyzer (YSI Life Sciences, Yellow Springs, OH, USA). Plasma lipids were measured using a Polychem Chemistry Analyzer (PolyMedCo, Inc. Cortlandt Manor, NY, USA). Plasma leptin and adiponectin concentrations were assessed by radioimmunoassay (Millipore, St. Charles, MO, USA). Plasma concentrations of active GLP-1 were quantified with an electro-chemiluminescent immunoassay (Meso Scale Discovery, Rockville, MD, USA).
HOMA-IR assessed systemic IR and was calculated using the equation [fasting glucose (mmol/L) × fasting insulin (µU/mL)]/22.5 [22 (link)].
HOMA-IR assessed systemic IR and was calculated using the equation [fasting glucose (mmol/L) × fasting insulin (µU/mL)]/22.5 [22 (link)].
4
Plasma Glucose and Hormone Assays
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Plasma glucose was determined at bedside by the glucose oxidase method with an Analox glucose analyzer (Analox Instruments, Lunenburg, MA). Total GLP-1 was measured by radioimmunoassay (Millipore) after plasma ethanol extraction. The assay reacts 100% with GLP17–36, GLP19–36, and GLP17–37, but not with glucagon (0.2%), GLP-2 (<0.01%), or exendin (<0.01%). Gastric inhibitory peptide (GIP) was determined by ELISA (Millipore) and reacts 100% with GIP1–42 and GIP3–42 but not with GLP-1, GLP-2, oxyntomodulin, or glucagon. Plasma insulin and C-peptide were measured by radioimmunoassay (Millipore). All hormone and metabolite assays were performed at the Hormonal Core Laboratory at the Obesity Nutrition Research Center. Intra- and interassay coefficients of variance ranged from 3.4–7.4 and 4.4–7.4%, respectively. Lipids were assayed by Ortho Clinical Diagnostics Vitros Fusion 5.1 (Ortho Clinical Diagnostics, Rochester, NY).
5
Plasma Glucagon and Insulin Measurement
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Plasma glucagon from mice fasted for 16 h were measured using radioimmunoassay (Millipore, Watford, U.K.). Blood (200 µl) was removed by cardiac puncture from mice killed by cervical dislocation. Plasma was collected using high speed (2000 g, 5 min at 4°C) centrifugation in heparin-coated Microvette® tubes containing EDTA (Sarstedt, Leicester, U.K.) with added DPP IV inhibitor (100 µM; Millipore, Watford, UK). For measurement of plasma insulin following intraperitoneal injection of glucose, blood (100 µl) was removed from the tail vein at various times points and plasma collected as described above. Plasma glucagon and insulin was measured by radioimmunoassay (Millipore).
6
Plasma Biomarker Analysis Methodology
7
Cord Blood Hormones Measurement
8
Serum Leptin and Estradiol Analysis
9
Analyzing miR-132 Effects on Insulin Secretion
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The MIN6 cells were transfected on day 1 with Mock or pEZX-miR-132 plasmid expressing the mature form of miR-132 from a commercial vector (Gene Copoeia, Rockville, MD, USA). Two days post-transfection, the cells were incubated in growth serum-free media with or without 0.5 mM palmitate/0.5% BSA for 24 h. On day 3 post-transfection, the cells were pre-incubated for 1 h at 37 °C in buffer containing 120 mmol/l NaCl, 5 mmol/l KCl, 2 mmol/l CaCl2, 1 mmol/l MgCl2, 24 mmol/l NaHCO3, 0.1% (wt./vol.) BSA, 15 mmol/l HEPES, and pH 7.4 with 0 mM glucose (resting media). After pre-incubation, the cells were re-incubated either in resting or stimulating media (25 mM glucose) for a 1.5 h. The media were collected and the cells were harvested and lysed in acidified ethanol (75% [vol./vol.] ethanol and 0.55% [vol./vol.] HCl). The amount of intracellular and released insulin was measured using a radioimmunoassay (Merck Millipore, Billerica, MA, USA).
10
Measuring Plasma Glucose Metabolism
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Plasma samples were placed on ice, centrifuged at 4°C, separated, and stored at −20°C until assayed. Glucose concentrations were measured using a glucose oxidase method (Yellow Springs Instruments, Yellow Springs, OH, USA). Plasma insulin was measured using a chemiluminescence assay (Access Assay; Beckman Coulter, Chaska, MN, USA). Plasma glucagon and C-peptide were measured by Radio-Immunoassay (EMD Millipore, Billerica, MA, USA). Plasma [6,6-2H2] glucose and [1-13C] glucose enrichments were measured using gas chromatographic mass spectrometry (Thermoquest, San Jose, CA, USA) to simultaneously monitor the C-1 and C-2 and C-3 to C-6 fragments, as described by Beylot et al.23 (link) In addition, [6-3H] glucose specific activity was measured by liquid scintillation counting following deproteinization and passage over anion and cation exchange columns.22 (link)
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