Double antibody radioimmunoassay

Manufactured by Merck Group

Sourced in United States

The Double-antibody radioimmunoassay is a laboratory technique used for the quantitative measurement of specific molecules or analytes in a sample. It involves the use of two antibodies: a primary antibody that binds to the target analyte and a secondary antibody that binds to the primary antibody. The sample is incubated with the primary antibody, and then the secondary antibody is added, which forms a complex with the primary antibody. This complex is then measured using a radioactive label, allowing for the quantification of the target analyte.

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Lab products found in correlation

Protease inhibitor cocktail, by Merck Group (5 mentions) Ysi 2300, by Yellow Springs Instruments (4 mentions) Dipeptidyl peptidase 4 inhibitor, by Merck Group (3 mentions) Dpp4 inhibitor, by Merck Group (2 mentions) Bwb 800, by Tanita (1 mentions) Pefabloc, by Roche (1 mentions) Ysi 2300 glucose analyzer, by Yellow Springs Instruments (1 mentions) Enzyme linked immunosorbent assay, by Merck Group (1 mentions) Hi 14k, by Merck Group (1 mentions) Hcp 20k, by Merck Group (1 mentions) Radioimmunoassay, by Merck Group (1 mentions) Body fat analyzer, by Tanita (1 mentions) Ysi 2300 stat plus, by Yellow Springs Instruments (1 mentions) Au5800, by Beckman Coulter (1 mentions) Au680, by Beckman Coulter (1 mentions) Unicel dxc880i, by Beckman Coulter (1 mentions) V plex proinflammatory panel 1 human kit, by Mesoscale (1 mentions) Abx pentra glucose hk cp, by Horiba (1 mentions) Abx pentra triglycerides cp, by Horiba (1 mentions) Cobas fara, by Roche (1 mentions)

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Radioimmunoassay (111 citations) Double antibody (2 citations)

13 protocols using double antibody radioimmunoassay

Comprehensive Metabolic Biomarker Analysis

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All samples were collected on ice in tubes containing EDTA. Protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) with dipeptidyl peptidase-4 inhibitor (Millipore, Billerica, MA) was immediately added and the samples were centrifuged at 4°C, separated, and frozen at −80°C. Plasma glucose was determined in duplicate by the glucose oxidase method using an automated glucose analyzer (YSI 2300; Yellow Springs Instruments, Yellow Springs, OH). Plasma immunoreactive insulin, C-peptide, and glucagon were measured in duplicate by double-antibody radioimmunoassays (Millipore) at the Penn Diabetes Research Center (DRC). Active GLP-1 and GLP-2 and total GIP were measured in duplicate by ELISA (Millipore) at the Penn DRC and the Translational Core Laboratory of the Children’s Hospital of Philadelphia, respectively. Enrichment of 6,6²H₂ glucose was measured using gas chromatography–mass spectrometry at the Metabolic Tracer Resource of the Penn Institute for Diabetes, Obesity and Metabolism.

Vetter M.L., Wadden T.A., Teff K.L., Khan Z.F., Carvajal R., Ritter S., Moore R.H., Chittams J.L., Iagnocco A., Murayama K., Korus G., Williams N.N, & Rickels M.R. (2014). GLP-1 Plays a Limited Role in Improved Glycemia Shortly After Roux-en-Y Gastric Bypass: A Comparison With Intensive Lifestyle Modification. Diabetes, 64(2), 434-446.

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Ghrelin Levels and Surgical Outcomes

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Participants were assessed within 4 weeks prior to their surgery date (baseline) and at 6 months ± 2 weeks after surgery. Weight-stable controls were assessed at baseline and at 6 months ± 2 weeks later. On scan days, following an overnight fast, participants completed a urine pregnancy and drug screening test, were weighed in a hospital gown on a calibrated scale (BWB 800S, Tanita Corp, Japan), and practiced the fMRI task. Just prior to imaging, blood was drawn from the forearm vein for measurement of ghrelin. Protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO), with dipeptidyl peptidase-4 inhibitor (Millipore, Billerica, MA) and pefabloc (Roche, Indianapolis, IN), was immediately added, and the samples were centrifuged at 4°C, separated, and frozen at −80°C. Ghrelin assays were measured in duplicate by double-antibody radioimmunoassays (Millipore) at the Penn Diabetes Research Center.

Faulconbridge L.F., Ruparel K., Loughead J., Allison K.C., Hesson L.A., Fabricatore A.N., Rochette A., Ritter S., Hopson R.D., Sarwer D.B., Williams N.N., Geliebter A., Gur R.C, & Wadden T.A. (2016). Changes in neural responsivity to highly-palatable foods following Roux-en-Y gastric bypass, sleeve gastrectomy, or weight stability: An fMRI study. Obesity (Silver Spring, Md.), 24(5), 1054-1060.

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Plasma Biomarker Measurement Protocol

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Plasma glucose was measured in duplicate by the glucose oxidase method using an automated glucose analyzer (YSI 2300; Yellow Springs Instruments, Yellow Springs, OH). Other samples were collected on ice into tubes containing EDTA and protease inhibitor cocktail (and for MMTT samples, DPP4 inhibitor; Sigma-Aldrich, St. Louis, MO), centrifuged at 4°C, separated, and frozen at −80°C for subsequent analysis. Plasma insulin, C-peptide, proinsulin, and glucagon were measured in duplicate by double-antibody radioimmunoassays (Millipore, Billerica, MA). Plasma free fatty acid levels were measured in duplicate using enzymatic colorimetrics (Wako Chemicals, Richmond, VA). Active glucagon-like peptide 1 (GLP-1) and total gastric inhibitory polypeptide (GIP) were measured in duplicate by ELISA (Millipore).

Sheikh S., Gudipaty L., De Leon D.D., Hadjiliadis D., Kubrak C., Rosenfeld N.K., Nyirjesy S.C., Peleckis A.J., Malik S., Stefanovski D., Cuchel M., Rubenstein R.C., Kelly A, & Rickels M.R. (2016). Reduced β-Cell Secretory Capacity in Pancreatic-Insufficient, but Not Pancreatic-Sufficient, Cystic Fibrosis Despite Normal Glucose Tolerance. Diabetes, 66(1), 134-144.

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Lipid and Glucose Biomarker Assessment

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Lipid parameters were assessed using standard laboratory procedures. Plasma glucose was measured in duplicate by the glucose oxidase method using an automated YSI 2300 glucose analyzer (Yellow Springs Instruments, Yellow Springs, OH). Plasma insulin and C-peptide were measured in duplicate by double-antibody radioimmunoassays (Millipore, Billerica, MA). Samples from matched case subjects and normal control subjects were assayed simultaneously.

Rickels M.R., Goeser E.S., Fuller C., Lord C., Bowler A.M., Doliba N.M., Hegele R.A, & Cuchel M. (2014). Loss-of-Function Mutations in ABCA1 and Enhanced β-Cell Secretory Capacity in Young Adults. Diabetes, 64(1), 193-199.

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Comprehensive Metabolic Biomarker Profiling

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Plasma glucose was measured in duplicate by the glucose oxidase method using an automated glucose analyzer (YSI 2300; Yellow Springs Instruments, Yellow Springs, OH). Samples were collected on ice into tubes containing EDTA and protease inhibitor cocktail (and for MMTT samples, DPP4 inhibitor; Sigma-Aldrich, St. Louis, Missouri), centrifuged at 4°C, separated, and frozen at −80°C for subsequent analysis. Plasma insulin, C-peptide, proinsulin, and glucagon were measured in duplicate by double antibody radioimmunoassays (Millipore, Billerica, Massachusetts). Active glucagon-like peptide 1 (GLP-1) and total glucose-dependent insulinotropic polypeptide (GIP) were measured in duplicate by enzyme-linked immunosorbent assays (Millipore).

Nyirjesy S.C., Sheikh S., Hadjiliadis D., De Leon D.D., Peleckis A.J., Eiel J.N., Kubrak C., Stefanovski D., Rubenstein R.C., Rickels M.R, & Kelly A. (2018). β-Cell secretory defects are present in pancreatic insufficient cystic fibrosis with 1-hour oral glucose tolerance test glucose ≥155 mg/dL. Pediatric diabetes, 19(7), 1173-1182.

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Plasma Glucose and Hormone Assays

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Plasma glucose was measured in duplicate by the glucose oxidase method using an automated glucose analyzer (YSI 2300; Yellow Springs Instruments, Yellow Springs, OH, USA). Additional blood samples were collected into tubes on ice containing ethylenediamine tetra-acetate and protease inhibitor cocktail and, for the MMTT test, dipeptidyl peptidase-4 inhibitor (Sigma-Aldrich, St. Louis, MO, USA). Samples were centrifuged at 4°C, separated, and frozen at −80°C for subsequent analysis. Plasma insulin (Millipore Cat# HI-14 K, RRID: AB_2801577) and C-peptide (Millipore Cat# HCP-20 K, RRID: AB_2891151) were assayed in duplicate by double-antibody radioimmunoassay (Millipore, Billerica, MA, USA).

Sheikh S., Stefanovski D., Kilberg M.J., Hadjiliadis D., Rubenstein R.C., Rickels M.R, & Kelly A. (2024). Early-phase insulin secretion during mixed-meal tolerance testing predicts β-cell function and secretory capacity in cystic fibrosis. Frontiers in Endocrinology, 15, 1340346.

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Comprehensive Metabolic Profile Assessment

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On a day separate from the fMRI session, a frequently-sampled oral glucose tolerance test (OGTT) (details below), fasting blood draw, and anthropometric measures were performed including weight, percent fat mass, and total fat mass (measured with a Tanita Body Fat Analyzer) and height (measured with a stadiometer). At 8:00 a.m. after an overnight fast, an intravenous catheter was placed for blood samples during a 3-h frequently sampled OGTT using Glucola (dose: 1.75 gm/kg glucose, maximum dose 75 g). Plasma glucose and insulin were measured at −15, 0, 10, 20, 30, 60, 90, 120, 150, and 180 min. Glucose was measured using glucose oxidase (YSI, Yellow Springs, OH), and plasma insulin was measured using a double-antibody radioimmunoassay (Millipore). In addition, a fasting blood sample for leptin was obtained and measured by radioimmunoassay (Millipore). For assessment of insulin resistance, whole-body insulin sensitivity index (WBISI) was calculated using the following formula: WBISI = 10,000/√[(FPG × fasting plasma insulin) × (mean OGTT glucose concentration × mean OGTT insulin concentration)] (34 (link)). BMI was calculated as weight in kilograms divided by the square of height in meters. A urine pregnancy test was performed on all female subjects.

Jastreboff A.M., Lacadie C., Seo D., Kubat J., Van Name M.A., Giannini C., Savoye M., Constable R.T., Sherwin R.S., Caprio S, & Sinha R. (2014). Leptin Is Associated With Exaggerated Brain Reward and Emotion Responses to Food Images in Adolescent Obesity. Diabetes Care, 37(11), 3061-3068.

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Biochemical Assays for Metabolic Markers

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Details of biochemical assays were described previously (31 (link)). Briefly, blood glucose concentrations were determined by the glucose oxidase method using a glucose analyzer (YSI 2300 STAT Plus; Yellow Springs Instruments, Yellow Springs, OH). Plasma immunoreactive insulin levels were measured using a double-antibody radioimmunoassay (Millipore, St. Charles, MO) (32 (link)). Plasma AG and desacylated ghrelin (DAG) levels were measured using separate sensitive and specific two-site sandwich ELISAs (16 (link)). The sensitivity of the AG assay was 6.7 pg/mL with intra- and interassay coefficients of variation of ∼14 and 18% (16 (link)). The sensitivity of the DAG assay was 4.6 pg/mL with intra- and interassay coefficients of variations of ∼13 and 20% (16 (link)). Glucagon was measured by radioimmunoassay (Millipore Life Sciences, Billerica, MA), and serum concentrations of human growth hormone were measured by a sandwich immunoassay using the automated Immulite 2000 chemiluminescent assay system (Siemens, Bad Nauheim, Germany) (31 (link)). Plasma free fatty acids were measured using a specific colorimetric assay (Wako Diagnostics, Richmond, VA). All samples were assayed in duplicate, and specimens from the four studies in each participant were run in the same assay.

Tong J., Davis H.W., Summer S., Benoit S.C., Haque A., Bidlingmaier M., Tschöp M.H, & D’Alessio D. (2014). Acute Administration of Unacylated Ghrelin Has No Effect on Basal or Stimulated Insulin Secretion in Healthy Humans. Diabetes, 63(7), 2309-2319.

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Blood Biomarker Dynamics After Ketone Ingestion

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Venous blood was collected via peripheral phlebotomy before and 30 min after KE ingestion. Serum β-hydroxybutyrate (BHB) levels were then determined on freshly processed samples using a Beckman-Coulter analyzer (AU5800 or AU680; Beckman-Coulter, Brea, CA). The reportable range for BHB is 0.02 to 13.5 mmol/L, and the coefficient of variation through multilevel, internal quality control ranges from 0.8% to 2.6%.
Insulin, glucagon, and nonesterified fatty acid (NEFA) were analyzed in bulk at the Penn Radioimmunoassay and Biomarkers Core. Blood samples were collected into tubes on ice containing ethylenediamine tetra-acetic acid and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Samples were centrifuged at 4°C, separated, and frozen at −80°C for subsequent analysis. NEFA levels (range, 0.125–2.9 mmol/L) were measured in duplicate using enzymatic colorimetry (Wako Chemicals, Richmond, VA). Plasma insulin (range, 2–100 mIU/mL) and glucagon (range, 20–400 pg/mL) were measured in duplicate using double-antibody radioimmunoassay (Millipore, Billerica, MA).

Selvaraj S., Hu R., Vidula M.K., Dugyala S., Tierney A., Ky B., Margulies K.B., Shah S.H., Kelly D.P, & Bravo P.E. (2021). Acute Echocardiographic Effects of Exogenous Ketone Administration in Healthy Participants. Journal of the American Society of Echocardiography : official publication of the American Society of Echocardiography, 35(3), 305-311.

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Fasting Plasma Biomarkers Assessment

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Participants had blood drawn after an overnight fast. Plasma glucose was measured by glucose oxidase method (YSI, Yellow Springs, OH) and insulin by double antibody radioimmunoassay (Millipore, St. Charles, MO). Plasma triglycerides and cholesterol were analyzed by enzymatic methods (Unicel DxC880i; Beckman Coulter, Inc., Brea, CA). HDL-C concentration was measured in the supernatant after precipitation with dextran sulfate. LDL-C concentration was computed using the Friedewald equation.[22 (link)]

Oursler K.K., Sorkin J.D., Ryan A.S, & Katzel L.I. (2018). A pilot randomized aerobic exercise trial in older HIV-infected men: Insights into strategies for successful aging with HIV. PLoS ONE, 13(6), e0198855.

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