Human E-Selectin/CD62E DuoSet ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human E-Selectin/CD62E. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.
Product Features
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Economical alternative to complete kits
Kit Content
- Capture Antibody
- Detection Antibody
- Recombinant Standard
- Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)
Other Reagents Required
DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.
The components listed above may be purchased separately:
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4
Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990)
Plate Sealers: ELISA Plate Sealers (Catalog # DY992)
Scientific Data
Product Datasheets
Preparation and Storage
Background: E-Selectin/CD62E
The Selectin family is comprised of three members, E-Selectin, L-Selectin, and P-Selectin. E-Selectin [endothelial leukocyte adhesion molecule-1 (ELAM-1), CD62E] is transiently expressed on vascular endothelial cells in response to IL-1 beta and TNF-alpha. The human and rat proteins share approximately 67% amino acid sequence identity. The mouse and rat proteins share approximately 78% amino acid sequence identity. L-Selectin (Leukocyte Selectin, LAM-1, LECAM-1, LECCAM-1, TQ1, Leu-8, MEL-14 antigen, DREG, lymph node homing receptor, CD62L) is expressed constitutively on a wide variety of leukocytes. Two forms of L-Selectin have been reported, apparently arising as a result of post-translational modifications. Human and mouse L-Selectin share 76% amino acid sequence identity. Human P-Selectin (GMP-140, LECAM-3, PADGEM, CD62P) is expressed by activated platelets and endothelial cells. The extracellular domains of human and mouse P-Selectin share approximately 73% amino acid sequence identity.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
- Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
- Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
- Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Repeat the aspiration/wash as in step 2.
- Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Citations for Human E-Selectin/CD62E DuoSet ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
11
Citations: Showing 1 - 10
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TNF Induces Laminin-332-Encoding Genes in Endothelial Cells and Laminin-332 Promotes an Atherogenic Endothelial Phenotype
Authors: Hayderi, A;Zegeye, MM;Meydan, S;Sirsjö, A;Kumawat, AK;Ljungberg, LU;
International journal of molecular sciences
Species: Human
Sample Types: Cell Culture Supernates, Cell Lysates
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Predictors of HIV rebound differ by timing of antiretroviral therapy initiation
Authors: Li, JZ;Melberg, M;Kittilson, A;Abdel-Mohsen, M;Li, Y;Aga, E;Bosch, RJ;Wonderlich, ER;Kinslow, J;Giron, LB;Di Germanio, C;Pilkinton, M;MacLaren, L;Keefer, M;Fox, L;Barr, L;Acosta, E;Ananworanich, J;Coombs, R;Mellors, J;Deeks, S;Gandhi, RT;Busch, M;Landay, A;Macatangay, B;Smith, DM;AIDS Clinical Trials Group A5345 Study Team, ;
JCI insight
Species: Human
Sample Types: Plasma
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Immuno-Modulatory Effects of Dexamethasone in Severe COVID-19-A Swedish Cohort Study
Authors: S Asif, R Frithiof, A Larsson, S Franzén, SB Anderberg, B Kristensen, M Hultström, M Lipcsey
Biomedicines, 2023-01-09;11(1):.
Species: Human
Sample Types: Plasma
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SARS-CoV-2 Spike Protein S1-Mediated Endothelial Injury and Pro-Inflammatory State Is Amplified by Dihydrotestosterone and Prevented by Mineralocorticoid Antagonism
Authors: N Kumar, Y Zuo, S Yalavarthi, KL Hunker, JS Knight, Y Kanthi, AT Obi, SK Ganesh
Viruses, 2021-11-03;13(11):.
Species: Human
Sample Types: Serum
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Complement activation is associated with poor outcome after out-of-hospital cardiac arrest
Authors: V Chaban, ER Nakstad, H Stær-Jense, C Schjalm, I Seljeflot, J Vaage, C Lundqvist, JŠ Benth, K Sunde, TE Mollnes, GØ Andersen, SE Pischke
Resuscitation, 2021-06-11;0(0):.
Species: Human
Sample Types: Plasma
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C13 Megastigmane Derivatives From Epipremnum pinnatum: beta-Damascenone Inhibits the Expression of Pro-Inflammatory Cytokines and Leukocyte Adhesion Molecules as Well as NF-kappaB Signaling
Authors: SP Pan, T Pirker, O Kunert, N Kretschmer, S Hummelbrun, SL Latkolik, J Rappai, VM Dirsch, V Bochkov, R Bauer
Front Pharmacol, 2019-11-28;10(0):1351.
Species: Human
Sample Types: Cell Culture Supernates
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Blood inflammatory and endothelial markers in women with von Willebrand disease
Authors: I Govorov, K Bremme, A Larsson, M Holmström, E Komlichenk, R Chaireti, M Mints
PLoS ONE, 2019-01-10;14(1):e0210544.
Species: Human
Sample Types: Plasma
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Early-onset preeclampsia predisposes to preclinical diastolic left ventricular dysfunction in the fifth decade of life: An observational study
Authors: A Bokslag, C Franssen, LJ Alma, I Kovacevic, FV Kesteren, PW Teunissen, O Kamp, W Ganzevoort, PL Hordijk, CJM Groot, WJ Paulus
PLoS ONE, 2018-06-12;13(6):e0198908.
Species: Human
Sample Types: Plasma
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Serum Soluble Vascular Cell Adhesion Molecule-1 Overexpression Is a Disease Marker in Patients with First-Time Diagnosed Antinuclear Antibodies: A Prospective, Observational Pilot Study
Authors: M Oleszowsky, MF Seidel
Biomed Res Int, 2018-02-01;2018(0):8286067.
Species: Human
Sample Types: Serum
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Serum retinol-binding protein-induced endothelial inflammation is mediated through the activation of toll-like receptor 4
Authors: M Du, A Martin, F Hays, J Johnson, RA Farjo, KM Farjo
Mol. Vis., 2017-03-31;23(0):185-197.
Species: Human
Sample Types: Cell Culture Supernates
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Suspension microarrays for the identification of the response patterns in hyperinflammatory diseases.
Authors: Hsu HY, Wittemann S, Schneider EM, Weiss M, Joos TO
Med Eng Phys, 2008-03-03;30(8):976-83.
Species: Human
Sample Types: Plasma
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We run human serum at a 1:20 dilution. It gave a perfect result with high precision. We recommand this ELISA kit.
We run human plasma samples at a 1:25 dilution in this assay. The assay performed excellent with high precision
