Reagents and kits were purchased from Sigma, except for the following: [1‐14C]‐palmitic acid (PerkinElmer Life Sciences, Santa Clara, CA, USA), liquid scintillation cocktail (LSC, PerkinElmer Life Sciences), [carboxyl‐14C]‐triolein (PerkinElmer Life Sciences), Man–Rogosa–Sharpe (MRS, Becton Dickinson, NJ, USA), orlistat (Xenical, Roche, Basel, Switzerland), EnzyChrom™ Free Fatty Acid Assay Kit (BioAssay Systems, Hayward, CA, USA). The sterilizable 384‐well plate and 384‐pin replicator were from Nunc. The membrane semidry system was from Bio‐Rad, and the X‐ray film was from Kodak. The gel‐pro analyzer software was from Media cybernetics. The magnetic resonance imaging (MRI) images were obtained with a Bruker Biospec 47/40 4.7‐Tesla instrument (Bruker, Billerica, MA, USA) and analyzed with image j software (NIH, Bethesda, MD, USA). The serum was analyzed with a Rat/Mouse ELISA kit (LINCO research, St. Charles, MO, USA), a Leptin ELISA kit (R&D System, Minneapolis, MN, USA), a blood glucose meter (Accu‐Chek, Roche) and Cholesterol ELISA kits (Asan Pharmaceutical, Seoul, Korea).
Leptin elisa kit
Manufactured by R&D Systems
Sourced in United States
The Leptin ELISA kit is a quantitative sandwich immunoassay designed for the measurement of leptin levels in human, mouse, and rat samples. The kit utilizes a specific anti-leptin antibody coated on a microplate to capture leptin in the samples. After a series of washes, a second anti-leptin antibody conjugated to horseradish peroxidase is added to detect the captured leptin. The resulting color change is measured and used to calculate the leptin concentration in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Rat insulin elisa kit, by Mercodia (2 mentions)
384 well plate, by Thermo Fisher Scientific (1 mentions)
X ray film, by Kodak (1 mentions)
Accu chek, by Roche (1 mentions)
Gel pro analyzer software, by Media Cybernetics (1 mentions)
Enzychrom free fatty acid assay kit, by BioAssay Systems (1 mentions)
Orlistat, by Roche (1 mentions)
1 14c palmitic acid, by PerkinElmer (1 mentions)
Automated analyzer system, by Roche (1 mentions)
D glucose, by Merck Group (1 mentions)
Insulin elisa kit, by Crystal Chem (1 mentions)
Adiponectin elisa kit, by R&D Systems (1 mentions)
Au680, by Beckman Coulter (1 mentions)
Corticosterone elisa kit, by Abcam (1 mentions)
Rat mouse insulin, by Merck Group (1 mentions)
Dkk1 elisa kit, by R&D Systems (1 mentions)
11 protocols using leptin elisa kit
1
Fatty acid metabolism in rodents
2
Biomarker Quantification in Serum Samples
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Glucose, cholesterol, and triglycerides were measured in the biochemistry lab of KAUH using serum samples stored at −80 °C. These measurements were made using an automated analyzer system (Roche Diagnostics, Mannheim, Germany). Leptin was measured using an enzyme-linked immunosorbent assay (ELISA) as described in Saadeh et al. [35 (link)]. Leptin ELISA kit was purchased from R&D Systems, Inc. (cat no. DY398; Minneapolis, MN, USA). Homeostasis model assessment-insulin resistance indices (HOMA-IR) were calculated using the following formula: (fasting insulin (μIU/L) × fasting glucose (mg/dL))/405.
3
Oral Glucose Tolerance Test in Obese Mice
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An oral glucose tolerance test (OGTT) was performed after four hours fasting in the lean C57BL/6 ad libitum (n = 6) and the DIO group (n = 6) 10 weeks after the beginning of the HFD exposure. A basal blood sample was taken by a tail cut, followed by a 20% D-glucose oral gavage bolus (1 g/kg, D-glucose was obtained from Sigma, St Louis, USA). Blood samples were taken at 5, 10, 15, 30, 60 and 120 minutes after the glucose bolus. The area under the curve of plasma glucose levels was calculated for each animal, DIO animals with a statistical significant higher area under the curve compared to the lean ad libitum animals were considered for further gene expression analysis. Plasma glucose, insulin (insulin ELISA kit, Crystal Chem, IL, USA) and leptin (leptin ELISA kit, R&D Systems, MN, USA) were also measured. The HOMA index was calculated using the following formula: (mM glucose value*μunits/ml insulin value)/22.5.
4
Cytokine and Leptin Quantification
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The levels of plasma cytokines (TNF-α, IL-6 and IL-1β) and leptin were measured via cytokine OptEIA™ kit (BD Biosciences) and leptin ELISA kit (R&D System), respectively, according to the manufacturers' instructions. The optical density was determined using a microplate reader set to 450 nm.
5
Quantifying Serum Leptin and Adiponectin
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Serum levels of leptin and adiponectin were analysed using leptin ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) and adiponectin ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer’s protocols.
6
Plasma Biomarkers in Fasted Mice
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Mice were sacrificed immediately after the 24 h fast. Corticosterone was measured colorimetrically using the Abcam corticosterone ELISA kit (Cambridge, MA, USA) in 5 μL plasma. Free-fatty acids (FFAs) were measured colorimetrically in 10 μL of plasma using the Randox NEFA Assay (Antrim, Ireland) and a Beckman-Coulter AU680 (Brea, CA, USA) following manufactures instructions. Leptin was measured colorimetrically using the R&D Systems leptin ELISA kit (Minneapolis, MN, USA).
7
Quantifying Leptin and IFNα in Mice
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Leptin concentration was measured in mouse serum and BAL fluid. A leptin ELISA kit was purchased from R & D Systems (Minneapolis, MN), and the manufacturer’s directions were followed to complete the assay. A mouse IFNα ELISA kit was purchased from Cell Sciences (Canton, MA) to detect IFNα in BAL fluids. The manufacturer’s instructions were followed to complete the assay.
8
Serum Metabolic Biomarker Profiling
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Blood samples were obtained quickly after sacrifice by cardiac puncture, collected in tubes and centrifuged at 3000 r/min for 10 min. at 4°C. Serum was collected and stored at −80°C. Glucose was determined using a colorimetric commercial kit (Sigma-Aldrich).
Plasma insulin concentrations were measured in duplicate by a rat/mouse insulin enzyme-linked immunosorbent assay (ELISA) kit, using rat insulin as the standard (Millipore Co.Vimodrone, Italy) according to the manufacturer’s instructions. The intra-assay coefficient of variability (CV) was 1.17–3.22%, the inter-assay CV was 6.71–9.23%, and the sensitivity limit was 0.1 ng/mL.
Leptin concentrations were measured using a leptin ELISA kit (R&D Systems, UK) according to the manufacturer’s instructions. The intra-assay CV was 3.8–4.3%, the inter-assay CV was 5–7.6%, and the sensitivity limit was 22 pg/mL. Quantification of free fatty acids (FFA) was performed using a commercially available ELISA kit (Bioassay Tech. Lab., China) according to the manufacturer’s instructions. The intra-assay CV was <10%, inter-assay CV was <12%, sensitivity was 2.51 mM/L.
Plasma insulin concentrations were measured in duplicate by a rat/mouse insulin enzyme-linked immunosorbent assay (ELISA) kit, using rat insulin as the standard (Millipore Co.Vimodrone, Italy) according to the manufacturer’s instructions. The intra-assay coefficient of variability (CV) was 1.17–3.22%, the inter-assay CV was 6.71–9.23%, and the sensitivity limit was 0.1 ng/mL.
Leptin concentrations were measured using a leptin ELISA kit (R&D Systems, UK) according to the manufacturer’s instructions. The intra-assay CV was 3.8–4.3%, the inter-assay CV was 5–7.6%, and the sensitivity limit was 22 pg/mL. Quantification of free fatty acids (FFA) was performed using a commercially available ELISA kit (Bioassay Tech. Lab., China) according to the manufacturer’s instructions. The intra-assay CV was <10%, inter-assay CV was <12%, sensitivity was 2.51 mM/L.
9
Serum Biomarker Analysis in Animal Model
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Blood samples were collected from euthanized animals by cardiac puncture and centrifuged (5714 × g for 5 min) for serum separation. Serum insulin levels were determined using a rat insulin ELISA kit (Mercodia, Uppsala, Sweden). Serum non-esterified fatty acids (NEFA) levels were analyzed using a colorimetric kit (Randox Laboratories, Antrim, United Kingdom), while leptin was analyzed using a Leptin ELISA kit (R&D Systems, Minneapolis, USA). Serum levels of cholesterol, triacylglycerol, HDL cholesterol and LDL cholesterol were determined by colorimetry using the BT 3000 equipment from Wiener laboratories.
10
Biomarkers in Ankylosing Spondylitis Patients
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The baseline clinical characteristics were collected, including patient symptoms and history, body mass index (BMI), C-reactive protein (CRP), erythrocyte sedimentation ratio (ESR), HLA-B27 test using PCR amplification with sequence-specific primers (BioSewoom Inc., Seoul, Korea), and X-rays of the cervical and lumbar spine. The ESR and CRP levels were measured at the first visit of outpatient clinic. The serum estradiol (E2), Dickkopf-1 (Dkk1), and leptin levels were measured in all patients using a human estrogen (E) enzyme-linked immunosorbent assay (ELISA) kit (CUSABIO, College Park, MD, USA), Dkk1 ELISA kit (R&D Systems, Minneapolis, MN, USA), and leptin ELISA kit (R&D Systems). Blood samples were obtained once at a routine visit at the rheumatologic clinic during the enrollment period. For females, menstrual cycle information was inquired about in an interview before the blood test, which was performed only in the follicular phase. Additional information regarding the presence or history of uveitis or enthesitis, psoriasis, inflammatory bowel disease, and peripheral arthritis was obtained from medical records.
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