In the morning after an overnight fast, venous blood was drawn in tubes containing sodium citrate (3.8%) and glucose (136 mM) and centrifuged at 2400 rpm for 10 min to obtain plasma. The plasma was stored at − 80 °C until use. The measurement of ADAM10 levels in the plasma was performed using a sandwich ELISA kit (Cloud-Clone Corp., Houston, TX, USA) that contained adhered anti-human ADAM10 antibodies, which reacted with the ADAM10 present in the samples. Secondary antibodies conjugated to the alkaline phosphatase enzyme, supplied by the kit, were used to bind to the adhered proteins and, after adding substrate to the enzyme, the absorbance reading of the plates was performed on a plate reader at 450 nm wavelength (Labtec LT4000). The minimum concentration detectable by the kit is 28 pg/mL, with a detection range between 78 and 5000 pg/mL and an intra-assay coefficient of variation below 10% and inter-assay below 12%.
Sandwich elisa kit
Manufactured by Cloud-Clone
Sourced in United States
The Sandwich ELISA kits are laboratory equipment designed for the detection and quantification of specific proteins or antigens in a sample. These kits utilize a sandwich-based enzyme-linked immunosorbent assay (ELISA) format, which involves the capture of the target analyte between two antibodies. The kits provide a reliable and sensitive method for analyzing the presence and concentration of the desired protein or antigen in various sample types.
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9 protocols using sandwich elisa kit
1
Measuring ADAM10 Levels in Plasma
2
Plasma PRCP Levels in Acute Optic Neuritis
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The PRCP levels were measured in plasma samples from the 20 patients with idiopathic acute ON and 20 healthy controls with Snellen visual acuities of 20/20. Blood samples of the patients were obtained on the day of ON diagnosis. Intravenous high-dose steroid therapy according to the ONTT regimen was initiated after the blood sample collection [3 (link)]. All patients had no previous neurological diseases or newly occurring neurologic symptoms related to their visual disturbance.
The plasma PRCP levels were measured using a commercially available Sandwich ELISA kit (Cloud-clone Corp., Wuhan, China), according to the manufacturer’s instructions. The assays were run in 96-well plates using 100 μL of plasma (1:10 dilution). After incubation and washing steps, the biotinylated detection antibody, HRP conjugate, and TMB substrate were added sequentially. The reaction was stopped with the stop solution, and the optical density was read at 450 nm using a microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA). The PRCP concentrations (pg/mL) were calculated from the standard curve using a four-parameter logistic regression model. The assay’s detection range was 31.25–2000 pg/mL, with an intra-assay coefficient of variation (CV) of <8% and an inter-assay CV of <10%. All measurements were collected from duplicated assays.
The plasma PRCP levels were measured using a commercially available Sandwich ELISA kit (Cloud-clone Corp., Wuhan, China), according to the manufacturer’s instructions. The assays were run in 96-well plates using 100 μL of plasma (1:10 dilution). After incubation and washing steps, the biotinylated detection antibody, HRP conjugate, and TMB substrate were added sequentially. The reaction was stopped with the stop solution, and the optical density was read at 450 nm using a microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA). The PRCP concentrations (pg/mL) were calculated from the standard curve using a four-parameter logistic regression model. The assay’s detection range was 31.25–2000 pg/mL, with an intra-assay coefficient of variation (CV) of <8% and an inter-assay CV of <10%. All measurements were collected from duplicated assays.
3
Serum Klotho Quantification Protocol
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A tube with an EDTA anticoagulant in the morning was filled with 5 mL of peripheral venous blood. Within 30 minutes of collection, blood samples were centrifuged for 15 minutes at 3000 rpm. The samples were devoid of serum and kept at −70°C for analysis. A sandwich ELISA kit was used to assess the serum Klotho level (Cloud-Clone Corp., Houston, TX, USA). At 450 nm, a microplate reader was used to calculate absorbance values.
4
TRPM8 Protein Quantification
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TRPM8 was measured in duplicate using a sandwich ELISA kit (Cloud-Clone Corp., Katy, TX, USA) according to the manufacturer's instructions. The limit of detection was 0.082 ng/mL. Values below this level were scored as 0 ng/mL for statistical analysis.
5
Cytokine Profiling of miRNA-Modulated HD11 Cells
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HD11 cells were transfected with miRNA mimics or control miRNA, or MMD-specific siRNA or control siRNA. LPS-treated HD11 cells were used as a positive control. The cell culture supernatants were collected at 24 h after transfection or treatment. The concentrations of TNF-α, IL-1β, IL-6 and IL-12α in cell culture supernatants were quantified using sandwich ELISA kits (Cloud-Clone).
6
Hormonal Evaluation via Oral Glucose
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For hormonal evaluation, blood samples were collected from the right tail vein, which was dissected and cannulated using a 26-gauge cannula. Blood was drawn via the cannula at 0 and 30 min after oral glucose was given, using tubes containing 10 μl EDTA (Sigma-Aldrich, St. Louis, USA) and 4 μl DPP-4 inhibitor (DRG Instruments GmbH, Marburg, Germany). After centrifugation at 4000 rpm for 10 min at 4 °C, samples were snap frozen in liquid nitrogen and stored at −80 °C until analysis. Insulin, GLP-1 (7-36) and glucose-dependent insulinotropic peptide, GIP serum concentrations were assessed in duplicates by sandwich ELISA kits (Cloud-Clone Corp., USA).
7
Mixed Meal Challenge in Rats
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Twelve weeks after surgery, a 4 ml/kg, 13.9 kJ/ml mixed meal was administered to 12-h-fasted rats by oral gavage in each group. Subsequently, blood samples were obtained from the rat tails every 15 min for 120 min, added to EDTA tubes containing dipeptidyl peptidase 4 (DPP-4) inhibitor (10 μl/ml blood; Millipore), and centrifuged at 4000 × g for 15 min at 4 °C. The plasma was removed, frozen, and stored at − 80 °C. PYY3-36 was assessed by sandwich ELISA kits (Cloud-Clone Corp, USA). GLP-2 was measured using an ELISA kit (Abcam, Cambridge CB4 OFL, UK) according to the manufacturer’s instructions. Finally, the area under the curve (AUC) was calculated by the trapezoidal rule for every parameter to study.
8
Gastrointestinal Hormones in RYGB Rats
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Four weeks after surgery, a 4 ml/kg, 13.9 KJ/ml mixed meal was administered to the 12-h fasting rats by oral gavage. Blood samples obtained from the rat tails, every 15 min for 120 min, were added to EDTA tubes containing dipeptidyl peptidase-4 (DPP-4) inhibitor (10 μl/ml blood; Millipore) and centrifuged at 4000 × g for 15 min at 4 °C; the plasma was then removed and frozen at -80 °C. GLP-1 and GIP were assessed by sandwich ELISA kits (Cloud-Clone Corp, USA). PYY was measured using an ELISA kit (ALPCO Diagnostics, Salem, NH) according to the manufacturer's instructions. The area under the curve (AUC) was calculated by the trapezoidal rule for every parameter in the study.
Three groups of 12-h fasting RYGB rats were included in the PYY intravenous infusions assay 4 weeks after surgery. Rat PYY (3-36) (Bachem, Bubendorf, Switzerland) was dissolved in saline (0.9% NaCl). A dose of 0.6 pmol kg -1 min -1 or vehicle (saline) was infused for 10 min in the first and second groups, and oral mixed meal was administered in the third group. GLP-1 and GIP plasma levels were quantified as above.
Three groups of 12-h fasting RYGB rats were included in the PYY intravenous infusions assay 4 weeks after surgery. Rat PYY (3-36) (Bachem, Bubendorf, Switzerland) was dissolved in saline (0.9% NaCl). A dose of 0.6 pmol kg -1 min -1 or vehicle (saline) was infused for 10 min in the first and second groups, and oral mixed meal was administered in the third group. GLP-1 and GIP plasma levels were quantified as above.
9
CSF Biomarker Measurement Protocol
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We obtained 5-10 mL of CSF samples from the lumbar punctures of each study participant. Each sample was centrifuged at 4000×g for 10 min. Then, the supernatant was collected and immediately frozen in polypropylene tubes that were stored at -80 • C until assay. The biomarker assays were performed in duplicate by experienced technicians under blinded conditions, and the intra-and interassay coefficients of variation were <20%. The concentrations of Aβ42, t-tau, p-tau were measured using the Luminex xMAP platform (Luminex Corp, Austin, TX) with INNO-BIA AlzBio3 (Fujirebio Europe, Inc., Ghent, Belgium) as previously described [15] . The concentrations of NfL were measured using the R-PLEX Human Neurofilament L Antibody Set (Meso Scale Discovery, Rockville, MD). The concentrations of soluble NG2 and α-syn were measured using commercially available sandwich ELISA kits (Cloud Clone Corp. Katy, TX and Covance, Dedham, MA, respectively). We only used samples with hemoglobin (Hb) concentrations <1000 ng/mL for α-syn analysis because, as previously reported, blood contamination (Hb > 1000 ng/mL) might affect the level of CSF α-syn [4] .
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