To estimate the amount of BDNF in lysates obtained from the hippocampi and hemispheres, the ChemiKine Brain Derived Neurotrophic Factor, Sandwich ELISA (Millipore) test was applied according to the supplier’s instructions. After performing the Sandwich ELISA assay, the plates were read at 450 nm using a spectrophotometric plate reader Fluorostar Omega (BMG LabTech).
Sandwich elisa
Manufactured by Merck Group
Sourced in United States, Germany
Sandwich ELISA is a type of enzyme-linked immunosorbent assay (ELISA) used to detect and quantify specific proteins or antigens in a sample. It involves the use of two antibodies: a capture antibody that is immobilized on a solid surface and a detection antibody that is conjugated with an enzyme. The sample is added to the solid surface, and the target antigen, if present, is captured by the immobilized antibody. The detection antibody then binds to the captured antigen, and the enzymatic reaction is used to generate a measurable signal, which is proportional to the amount of the target antigen in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Fluorostar omega, by BMG Labtech (1 mentions)
Competitive enzyme immunoassay kit, by Cayman Chemical (1 mentions)
Hplc system, by Thermo Fisher Scientific (1 mentions)
Accu check, by Roche (1 mentions)
Radioimmunoassay, by Merck Group (1 mentions)
Abx pentra 400, by Horiba (1 mentions)
Cobas integra 400, by Roche (1 mentions)
Magpix milliplex kit, by Merck Group (1 mentions)
13 protocols using sandwich elisa
1
BDNF Quantification in Brain Lysates
2
Plasma Insulin Level Determination by ELISA
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The plasma insulin levels were determined by Sandwich ELISA (Millipore). The plasma samples were added to a microtiter plate coated with monoclonal mouse anti-rat insulin antibodies. After that, unbound material from the samples was washed away and the immobilized biotinylated antibodies were added. The microtiter plate had the substrate 3,3′,5,5′-tetramethybenzidine added. The enzyme activity was measured by a spectrophotometer (BioTek) with an absorbance at 450 nm, with a corrected wavelength at 590 nm. The plasma insulin concentration in the unknown samples was derived by interpolation from a reference curve generated in the same assay with reference standard of known concentration of rat insulin.
3
Metabolic Profile and Oxidative Stress Assessment
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Metabolic parameters were measured by an enzymatic colorimetric assay using a commercially available kit (Biotech, Bangkok, Thailand). Fasting plasma HDL, LDL, and insulin levels were measured using a sensitive, colorimetric and fluorometric assay (BioVision), Friedewald’s equation [24 (link)] and Sandwich ELISA (Millipore), respectively.
Insulin resistance was determined using the homeostasis model assessment (HOMA) [25 (link)]. A higher HOMA index indicates a greater degree of insulin resistance [13 (link)]. Total plasma testosterone and estradiol levels were determined by ELISA and a competitive enzyme immunoassay kit (Cayman Chemical Company, MI, USA), respectively.
Plasma and cardiac malondialdehyde (MDA) levels were determined using an HPLC system (Thermo Scientific) [18 (link)]. The concentrations of either plasma or cardiac thiobarbituric acid reactive substances (TBARS) were obtained from a standard curve, and reported as an MDA equivalent concentration [26 ].
Insulin resistance was determined using the homeostasis model assessment (HOMA) [25 (link)]. A higher HOMA index indicates a greater degree of insulin resistance [13 (link)]. Total plasma testosterone and estradiol levels were determined by ELISA and a competitive enzyme immunoassay kit (Cayman Chemical Company, MI, USA), respectively.
Plasma and cardiac malondialdehyde (MDA) levels were determined using an HPLC system (Thermo Scientific) [18 (link)]. The concentrations of either plasma or cardiac thiobarbituric acid reactive substances (TBARS) were obtained from a standard curve, and reported as an MDA equivalent concentration [26 ].
4
Intraperitoneal Glucose Tolerance Test
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An intraperitoneal glucose tolerance test (IPGTT) was performed at the end of the 4-week treatment period. Briefly, after 1 day of fasting, 50% dextrose (1.5 g/kg) was administered to mice. The blood glucose concentration was measured just before and at 30, 60, 90, and 120 min after dextrose administration using a glucose analyzer (Accu-Check; Roche Diagnostics, Basel, Switzerland). The area under the curve of glucose (AUCg) was calculated by trapezoidal estimation from the concentrations obtained in the IPGTT. The fasting serum insulin level was measured using a sandwich ELISA (Millipore Corporation, St. Charles, MO, USA). For determination of islet size, the numbers of beta cells were quantified in captured immunohistochemistry images for insulin detection (TDI Scope Eye, Version 3.6 for Windows; Seoul, Korea). Insulinpositive cells were quantified by counting cells in ~20 randomly selected non-overlapping islets for all eight mice in each group.
5
Metabolic and Hormonal Biomarkers Profiling
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Fasting plasma glucose, total cholesterol, and triglyceride concentrations were determined by an enzymatic colorimetric assay from a commercially available kit (Biotech, Bangkok, Thailand). Fasting plasma HDL was determined using a sensitive, colorimetric and fluorometric assay from a commercially available kit (BioVision), whereas plasma LDL was determined using Friedewald's equation (Friedewald et al. 1972) . Fasting plasma insulin level was determined by Sandwich ELISA (Millipore).
Insulin resistance was assessed using the homeostasis model assessment (HOMA) (Appleton et al. 2005) (link), which was calculated from fasting plasma insulin and fasting plasma glucose concentrations. A higher HOMA index indicates a higher degree of insulin resistance (Pratchayasakul et al. 2011) (link). Total plasma testosterone (ng/dL) was analyzed by ELISA technique at the Central Laboratory Service of Maharaj Nakorn Chiang Mai Hospital, Faculty of Medicine, Chiang Mai University.
Insulin resistance was assessed using the homeostasis model assessment (HOMA) (Appleton et al. 2005) (link), which was calculated from fasting plasma insulin and fasting plasma glucose concentrations. A higher HOMA index indicates a higher degree of insulin resistance (Pratchayasakul et al. 2011) (link). Total plasma testosterone (ng/dL) was analyzed by ELISA technique at the Central Laboratory Service of Maharaj Nakorn Chiang Mai Hospital, Faculty of Medicine, Chiang Mai University.
6
Metabolic and Hormonal Assessment Protocol
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Fasting plasma glucose, total cholesterol and triglyceride concentrations were determined by an enzymatic colorimetric assay from a detection kit (Biotech, Bangkok, Thailand). Fasting plasma HDL was determined using colorimetric and fluorometric assays from a detection kit (BioVision), whereas plasma LDL was determined using Friedewald's equation using the fasting plasma HDL values (Friedewald et al. 1972) . Fasting plasma insulin levels were determined using a sandwich ELISA (Millipore). Insulin resistance was assessed using the Homeostasis Model Assessment (HOMA) (Appleton et al. 2005) (link). A higher HOMA index indicates a higher degree of insulin resistance (Pratchayasakul et al. 2011) (link). Total plasma testosterone (ng/mL) was analyzed by ELISA at the Central Laboratory Service of Maharaj Nakorn Chiang Mai Hospital, Faculty of Medicine, Chiang Mai University.
7
Quantification of Metabolic Biomarkers
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Ghrelin (LOD: 50 pg mL–1) and GLP‐1 (LOD: 2 pm ) were determined by means of a sandwich ELISA (Merck Millipore, Germany), whereas serotonin was quantified by means of a competitive ELISA (LOD: 2.6 ng mL–1). Serotonin samples under this LOD were additionally analyzed using the serotonin high sensitive ELISA with an LOD of 0.39 pg per sample (both DLD Diagnostika, Hamburg, Germany). Insulin concentrations were assessed using a sandwich ELISA (LOD: 50 pg mL–1) obtained from IASON (Graz, Austria). Glucose concentrations were quantitated by a colorimetric assay, presenting an LOD of 0.23 mg dL–1 (Cayman Europe, Tallinn, Estonia).
8
Quantification of Plasma Biomarkers
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Total GLP‐1 (LOD: 2 pm , inter‐assay CV 8 ± 4.8%, intra‐assay CV 7.4 ± 1.1%) and glucagon (LOD: 2.5 pg mL−1, inter‐assay CV < 12%, intra‐assay CV < 10%) plasma concentrations were determined by means of a sandwich ELISA (Merck Millipore, Darmstadt, Germany, and Thermo–Fisher Scientific, Waltham, USA, respectively). Plasma glucose concentrations were quantitated by a colorimetric assay with an LOD of 0.23 mg dl−1 (inter‐assay CV 1.7%, intra‐assay CV 4.6%) (Cayman Europe, Tallinn, Estonia). Insulin concentrations in the plasma were assessed using sandwich ELISA (LOD: 50 pg mL−1, inter‐assay CV 2.6%, intra‐assay CV 5.99%) obtained from IASON (Graz, Austria).
9
Comprehensive Plasma and Serum Biomarker Analysis
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The analysis of all plasma and serum samples was performed in duplicate using the methods outlined below. Appropriate radioimmunoassays (EMD Millipore, Billerica, U.S.) were used to determine the concentration of total ghrelin, PYY, insulin, leptin, and adiponectin. GLP-1 concentration was determined using sandwich ELISA (EMD Millipore, Billerica, U.S.), comprising amide acid GLP-1 7-36 and 7-37. The glucose concentration was assessed using the glucose oxidase enzymatic method (YSI Inc., Yellow Springs, Ohio, USA).
The concentration of FFA, TAG, HDL-C, LDL-C and total cholesterol was determined using spectrophotometry (ABX Pentra 400, Horiba Medical, Montpellier, France). Adrenaline and noradrenaline concentrations were determined by extraction of both from the plasma (HPLC – ECD; Glutathione preservative E4378, Sigma-Aldrich Company Ltd., Dosert, U.K.), followed by high performance liquid chromatography with electrochemical detection [34] (link). The intra-assay coefficient of variation (CV) for leptin, FFA, TAG, HDL-C, LDL-C and total cholesterol (analyzed in a single run) were 1.0%, 1.5%, 2.9%, 1.3%, 1.0% and 4.6%, respectively. The inter-assay CV for total ghrelin, PYY, GLP-1, insulin, plasma glucose, adiponectin, adrenaline and noradrenaline (analyzed in multiple runs) were 9.1%, 6.7%, 4.9%, 7.4%, 2.3%, 8.6%, 11.8% and 17.2%, respectively.
The concentration of FFA, TAG, HDL-C, LDL-C and total cholesterol was determined using spectrophotometry (ABX Pentra 400, Horiba Medical, Montpellier, France). Adrenaline and noradrenaline concentrations were determined by extraction of both from the plasma (HPLC – ECD; Glutathione preservative E4378, Sigma-Aldrich Company Ltd., Dosert, U.K.), followed by high performance liquid chromatography with electrochemical detection [34] (link). The intra-assay coefficient of variation (CV) for leptin, FFA, TAG, HDL-C, LDL-C and total cholesterol (analyzed in a single run) were 1.0%, 1.5%, 2.9%, 1.3%, 1.0% and 4.6%, respectively. The inter-assay CV for total ghrelin, PYY, GLP-1, insulin, plasma glucose, adiponectin, adrenaline and noradrenaline (analyzed in multiple runs) were 9.1%, 6.7%, 4.9%, 7.4%, 2.3%, 8.6%, 11.8% and 17.2%, respectively.
10
Biochemical Markers of Liver Health
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Biochemical blood tests were carried out to evaluate liver functions in the form of serum transaminases (SGOT and SGPT), alkaline phosphatase (ALP), gamma glutamate (GGT), total bilirubin, as well as albumin. Serum SGPT, SGOT, GGT and ALP levels were measured by the kinetic methods according to the recommendations of the Committee on the Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology (1974 & 1976). Serum albumin was measured by colorimetric methods.
Patients were diagnosed as having hepatitis C by detecting HCV antibodies using a third generation enzyme immunoassay, ELISA.
In CHC patients, the baseline HCV RNA load were measured using a quantitative polymerase chain reaction (PCR) assay according to the available technique (Applied Biosystem, USA), with detection limit of 30 IU/ml.
GDF15 was measured in serum using a specific ELISA kit (Cat# KT-16893, R&D Systems, Kamiya Biomedical Co., Seattle, WA), according to the manufacturer’s recommended protocol.
Determination of serum Alfa fetoprotein: Serum samples were analyzed for Alfa fetoprotein using a commercially available sandwich ELISA kit (Cat# DAFP00, R&D Systems, USA & Canada).
Determination of serum Ferritin: serum samples were analyzed for Ferritin using a commercially available sandwich ELISA obtained from Sigma-Aldrich).
Patients were diagnosed as having hepatitis C by detecting HCV antibodies using a third generation enzyme immunoassay, ELISA.
In CHC patients, the baseline HCV RNA load were measured using a quantitative polymerase chain reaction (PCR) assay according to the available technique (Applied Biosystem, USA), with detection limit of 30 IU/ml.
GDF15 was measured in serum using a specific ELISA kit (Cat# KT-16893, R&D Systems, Kamiya Biomedical Co., Seattle, WA), according to the manufacturer’s recommended protocol.
Determination of serum Alfa fetoprotein: Serum samples were analyzed for Alfa fetoprotein using a commercially available sandwich ELISA kit (Cat# DAFP00, R&D Systems, USA & Canada).
Determination of serum Ferritin: serum samples were analyzed for Ferritin using a commercially available sandwich ELISA obtained from Sigma-Aldrich).
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