Human S100A8/S100A9 Heterodimer 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 S100 Calcium Binding Protein A8/S100 Calcium Binding Protein A9 (S100A8/S100A9 Heterodimer). The suggested diluent is suitable for the analysis of most cell culture supernate, serum, plasma, saliva, human milk, urine, and fecal samples. Diluents for complex matrices, such as serum, plasma, saliva, human milk, urine, and feces, 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
Wash Buffer: (Catalog # WA126), or equivalent
Reagent Diluent*
Blocking Buffer*
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), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent
*For the Reagent Diluent and Blocking Buffer recommended for a specific DuoSet ELISA Development Kit, please see the product.
Scientific Data
Product Datasheets
Preparation and Storage
Background: S100A8/S100A9 Heterodimer
S100A8 (also MRP8 and calgranulin A) is a 10 kDa member of the S100 family, EF-hand superfamily of Ca-binding proteins. It is produced by neutrophils and monocytes, and forms Ca2+-dependent heterodimer/heterotetramer complexes (termed calprotectin) with S100A9. It functions both intracellularly and extracellularly, where it binds to RAGE and CD36. Human S100A8 is 93 amino acids (aa) in length. It contains two EF-hand motifs (aa 12 - 47 and 46 - 81) and one high-affinity Ca2+-binding site (aa 59 - 70). There may be one splice form that shows a 15 aa substitution for the C-terminal 14 amino acids. Although mouse S100A8 is cleaved by MMP-2 after Asn21, it is unclear if human S100A8 is susceptible. Full-length human S100A8 is 57% and 74% aa identical to mouse and canine S100A8, respectively.
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 Block Buffer 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 with NGS, 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 S100A8/S100A9 Heterodimer 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.
19
Citations: Showing 1 - 10
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The assembly of neutrophil inflammasomes during COVID-19 is mediated by type I interferons
Authors: Cabrera, LE;Jokiranta, ST;Mäki, S;Miettinen, S;Kant, R;Kareinen, L;Sironen, T;Pietilä, JP;Kantele, A;Kekäläinen, E;Lindgren, H;Mattila, P;Kipar, A;Vapalahti, O;Strandin, T;
PLoS pathogens
Species: Human
Sample Types: Serum
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Elevated neutrophil extracellular traps in systemic sclerosis-associated vasculopathy and suppression by a synthetic prostacyclin analog
Authors: Kortam, N;Liang, W;Shiple, C;Huang, S;Gedert, R;Clair, JS;Sarosh, C;Foster, C;Tsou, PS;Varga, J;Knight, JS;Khanna, D;Ali, RA;
Arthritis research & therapy
Species: Human
Sample Types: Plasma
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The Elevated Inflammatory Status of Neutrophils Is Related to In-Hospital Complications in Patients with Acute Coronary Syndrome and Has Important Prognosis Value for Diabetic Patients
Authors: Barbu, E;Mihaila, AC;Gan, AM;Ciortan, L;Macarie, RD;Tucureanu, MM;Filippi, A;Stoenescu, AI;Petrea, SV;Simionescu, M;Balanescu, SM;Butoi, E;
International journal of molecular sciences
Species: Human
Sample Types: Serum
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Using the Traditional Ex Vivo Whole Blood Model to Discriminate Bacteria by Their Inducible Host Responses
Authors: Chick, HM;Rees, ME;Lewis, ML;Williams, LK;Bodger, O;Harris, LG;Rushton, S;Wilkinson, TS;
Biomedicines
Species: Human
Sample Types: Whole Blood
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Viral nanoparticle vaccines against S100A9 reduce lung tumor seeding and metastasis
Authors: Chung, YH;Ortega-Rivera, OA;Volckaert, BA;Jung, E;Zhao, Z;Steinmetz, NF;
Proceedings of the National Academy of Sciences of the United States of America
Species: Mouse
Sample Types: Serum, Tissue Homogenates
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Ginger intake suppresses neutrophil extracellular trap formation in autoimmune mice and healthy humans
Authors: Ali, RA;Minarchick, VC;Zahavi, M;Rysenga, CE;Sturm, KA;Hoy, CK;Sarosh, C;Knight, JS;Demoruelle, MK;
JCI insight
Species: Human
Sample Types: Plasma
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Chronic inflammation, neutrophil activity, and autoreactivity splits long COVID
Authors: Woodruff, MC;Bonham, KS;Anam, FA;Walker, TA;Faliti, CE;Ishii, Y;Kaminski, CY;Ruunstrom, MC;Cooper, KR;Truong, AD;Dixit, AN;Han, JE;Ramonell, RP;Haddad, NS;Rudolph, ME;Yalavarthi, S;Betin, V;Natoli, T;Navaz, S;Jenks, SA;Zuo, Y;Knight, JS;Khosroshahi, A;Lee, FE;Sanz, I;
Nature communications
Species: Human
Sample Types: Plasma
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Single cell transcriptomics identifies distinct profiles in pediatric acute respiratory distress syndrome
Authors: Flerlage, T;Crawford, JC;Allen, EK;Severns, D;Tan, S;Surman, S;Ridout, G;Novak, T;Randolph, A;West, AN;Thomas, PG;
Nature communications
Species: Human
Sample Types: Tracheal Aspirate
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SCCA1/SERPINB3 promotes suppressive immune environment via STAT-dependent chemokine production, blunting the therapy-induced T cell responses
Authors: L Chen, V Shi, S Wang, R Freeman, F Ruiz, K Jayachandr, J Zhang, P Cosper, L Sun, CJ Luke, C Spina, PW Grigsby, JK Schwarz, S Markovina
bioRxiv : the preprint server for biology, 2023-02-03;0(0):.
Species: Human
Sample Types: Cell Culture Supernates
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The Effect of Nutritional Intervention with Lactoferrin, Galactooligosacharides and Vitamin D on the Gut Microbiota Composition of Healthy Elderly Women
Authors: P Konstanti, M van Splunt, E van den Br, C Belzer, A Nauta, RJJ van Neerve, H Smidt
Nutrients, 2022-06-14;14(12):.
Species: Human
Sample Types: Fecal Supernates
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Antibody microarray analysis of amniotic fluid proteomes in women with cervical insufficiency and short cervix, and their association with pregnancy latency length
Authors: S Hong, KH Park, YE Lee, JE Lee, YM Kim, E Joo, I Cho
PLoS ONE, 2022-02-07;17(2):e0263586.
Species: Human
Sample Types: Amniotic Fluid
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Circulating Interleukin-6 and CD16 positive monocytes increase following angioplasty of an arteriovenous fistula
Authors: S Hakki, EJ Robinson, MG Robson
Scientific Reports, 2022-01-26;12(1):1427.
Species: Human
Sample Types: Plasma
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Pancreatic ductal deletion of S100A9 alleviates acute pancreatitis by targeting VNN1-mediated ROS release to inhibit NLRP3 activation
Authors: H Xiang, F Guo, X Tao, Q Zhou, S Xia, D Deng, L Li, D Shang
Theranostics, 2021-03-04;11(9):4467-4482.
Species: Human
Sample Types: Cell Culture Supernates
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Plasma tissue plasminogen activator and plasminogen activator inhibitor-1 in hospitalized COVID-19 patients
Authors: Y Zuo, M Warnock, A Harbaugh, S Yalavarthi, K Gockman, M Zuo, JA Madison, JS Knight, Y Kanthi, DA Lawrence
Scientific Reports, 2021-01-15;11(1):1580.
Species: Human
Sample Types: Plasma
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A protein microarray analysis of amniotic fluid proteins for the prediction of spontaneous preterm delivery in women with preterm premature rupture of membranes at 23 to 30 weeks of gestation
Authors: HJ Kim, KH Park, YM Kim, E Joo, K Ahn, S Shin
PLoS ONE, 2020-12-31;15(12):e0244720.
Species: Human
Sample Types: Amniotic Fluid
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Prothrombotic antiphospholipid antibodies in COVID-19
Authors: Y Zuo, SK Estes, AA Gandhi, S Yalavarthi, RA Ali, H Shi, G Sule, K Gockman, JA Madison, M Zuo, W Woodard, SP Lezak, NL Lugogo, Y Kanthi, JS Knight
medRxiv, 2020-09-15;0(0):.
Species: Human
Sample Types: Serum
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Neutrophil calprotectin identifies severe pulmonary disease in COVID-19
Authors: H Shi, Y Zuo, S Yalavarthi, K Gockman, M Zuo, JA Madison, CN Blair, W Woodard, SP Lezak, NL Lugogo, RJ Woods, C Lood, JS Knight, Y Kanthi
medRxiv, 2020-07-15;0(0):.
Species: Human
Sample Types: Serum
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Immune and Inflammatory Proteins in Cord Blood as Predictive Biomarkers of Retinopathy of Prematurity in Preterm Infants
Authors: YJ Park, SJ Woo, YM Kim, S Hong, YE Lee, KH Park
Invest. Ophthalmol. Vis. Sci., 2019-09-03;60(12):3813-3820.
Species: Human
Sample Types: Plasma
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CXCL4 assembles DNA into liquid crystalline complexes to amplify TLR9-mediated interferon-? production in systemic sclerosis
Authors: R Lande, EY Lee, R Palazzo, B Marinari, I Pietrafort, GS Santos, Y Mattenberg, F Spadaro, K Stefananto, N Iannace, AM Dufour, M Falchi, M Bianco, E Botti, L Bianchi, M Alvarez, V Riccieri, ME Truchetet, G C L Wong, C Chizzolini, L Frasca
Nat Commun, 2019-05-01;10(1):1731.
Species: Human
Sample Types: Plasma
FAQs
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For the Human S100A8/S100A9 Heterodimer DuoSet ELISA, Catalog # DY8226-05, can Tween 20 be substituted for Brij® 35 in the Reagent Diluent?
The 50 mM Tris, 10 mM CaCl2, 0.15 M NaCl, 0.05% Brij® 35, pH 7.45-7.5 that we recommend as a reagent diluent for this kit is unique, which means that our lab would have found that more traditional reagent diluents do not perform optimally. It is certainly possible that you could potentially substitute a different detergent (such as Tween) with viable results, but the performance of this different formulation of the reagent diluent would need to be empirically determined on your end.
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