Human CXCL5/ENA-78 DuoSet ELISA

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Human CXCL5 / ENA-78 ELISA Standard Curve
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Product Details
Citations (19)
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Human CXCL5/ENA-78 DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
96-well strip plate
Sample Volume Required
100 µL
Assay Range
15.6 - 1,000 pg/mL
Sufficient Materials
For five or fifteen 96-well plates*
Please see the product datasheet

* 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 CXCL5/ENA-78. 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

Human CXCL5 / ENA-78 ELISA Standard Curve

Product Datasheets

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Preparation and Storage

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: CXCL5/ENA-78

CXCL5/Epithelial Cell-derived Neutrophil-activating Peptide (ENA-78), is a member of the CXC subfamily of chemokines. Full-length CXCL5/ENA-78 is 114 amino acids (aa) in length with a predicted molecular weight of 12 kDa. Following the removal of the signal peptide, bioactive CXCL5/ENA-78 is 78 aa in length. CXCL5/ENA-78 can be N-terminally cleaved by Cathepsin G and Chymotrypsin to CXCL5/ENA-74 (74 aa) and CXCL5/ENA-70 (70 aa), which show increased potency relative to CXCL5/ENA-78. While murine LIX was thought to be an ortholog to human CXCL5/ENA-78, genome-wide analysis and a consensus in the field suggests that human CXCL5/ENA-78 does not have a true murine ortholog. CXCL5/ENA-78 is upregulated at sites of inflammation and is expressed by multiple hematopoietic cell types, fibroblasts, endothelial cells, vascular smooth muscle cells, and adipocytes. It acts as a chemoattractant for neutrophils, has angiogenic properties, and contributes to cancer progression.

Entrez Gene IDs:
6374 (Human)
Alternate Names:
chemokine (C-C motif) ligand 5; C-X-C motif chemokine 5; CXCL5; CXCL5/ENA-78; ENA78; ENA-78; ENA-78(1-78); Epithelial-derived neutrophil-activating protein 78; Neutrophil-activating peptide ENA-78; neutrophil-activating protein 78; SCYB5ENA78

Assay Procedure


Plate Preparation

  1. 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.
  2. 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.
  3. Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. 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.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. 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.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. 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.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. 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 CXCL5/ENA-78 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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  1. ADSCs stimulated by resistin promote breast cancer cell malignancy via CXCL5 in a breast cancer coculture model
    Authors: YY Wang, AC Hung, YC Wu, S Lo, HD Chen, YK Chen, YC Hsieh, SC Hu, MF Hou, SF Yuan
    Scientific Reports, 2022-09-14;12(1):15437.
    Species: Human
    Sample Types: Cell Culture Supernates
  2. Pro-cancerogenic effects of spontaneous and drug-induced senescence of ovarian cancer cells in vitro and in vivo: a comparative analysis
    Authors: S Rutecki, P Szulc, M Paku?a, P Uruski, A Radziemski, E Naumowicz, R Moszy?ski, A Tykarski, J Miku?a-Pie, K Ksi??ek
    Journal of ovarian research, 2022-07-26;15(1):87.
    Species: Human
    Sample Types: Cell Culture Supernates
  3. Differential expression profile of CXC-receptor-2 ligands as potential biomarkers in pancreatic ductal adenocarcinoma
    Authors: S Saxena, C Molczyk, A Purohit, E Ehrhorn, P Goel, DR Prajapati, P Atri, S Kaur, PM Grandgenet, MA Hollingswo, SK Batra, RK Singh
    American journal of cancer research, 2022-01-15;12(1):68-90.
    Species: Human
    Sample Types: Cell Culture Supernates
  4. Patient Derived Colonoids as Drug Testing Platforms-Critical Importance of Oxygen Concentration
    Authors: HK Skovdahl, S Gopalakris, TD Svendsen, AVB Granlund, I Bakke, ZG Ginbot, S Thorsvik, A Flatberg, B Sporsheim, J Ostrop, TE Mollnes, AK Sandvik, T Bruland
    Frontiers in Pharmacology, 2021-05-13;12(0):679741.
    Species: Human
    Sample Types: Cell Culture Supernates
  5. Adipocytokines in Untreated Newly Diagnosed Rheumatoid Arthritis: Association with Circulating Chemokines and Markers of Inflammation
    Authors: GK Vasileiadi, AC Lundell, Y Zhang, K Andersson, I Gjertsson, A Rudin, C Maglio
    Biomolecules, 2021-02-21;11(2):.
    Species: Human
    Sample Types: Plasma
  6. Clinical Relevance of the Anti-inflammatory Effects of Roflumilast on Human Bronchus: Potentiation by a Long-Acting Beta-2-Agonist
    Authors: H Salvator, A Buenestado, M Brollo, E Naline, T Victoni, E Longchamp, H Tenor, S Grassin-De, P Devillier
    Frontiers in Pharmacology, 2020-12-08;11(0):598702.
    Species: Human
    Sample Types: Cell Culture Supernates
  7. Biofluid quantification of TWEAK/Fn14 axis in combination with a selected biomarker panel improves assessment of prostate cancer aggressiveness
    Authors: X Ruiz-Plaza, E Rodríguez-, M Alves, A Altuna-Coy, J Lozano-Bar, M Portero-Ot, JF García-Fon, S Martínez-G, J Segarra, MR Chacón
    J Transl Med, 2019-09-09;17(1):307.
    Species: Human
    Sample Types: Serum
  8. Resistance to lysosomotropic drugs used to treat kidney and breast cancers involves autophagy and inflammation and converges in inducing CXCL5
    Authors: S Giuliano, M Dufies, PD Ndiaye, J Viotti, D Borchielli, J Parola, V Vial, Y Cormerais, M Ohanna, V Imbert, E Chamorey, N Rioux-Lecl, A Savina, JM Ferrero, B Mograbi, G Pagès
    Theranostics, 2019-01-30;9(4):1181-1199.
    Species: Human
    Sample Types: Cell Culture Supernates
  9. Excessive neutrophil levels in the lung underlie the age-associated increase in influenza mortality
    Authors: U Kulkarni, RL Zemans, CA Smith, SC Wood, JC Deng, DR Goldstein
    Mucosal Immunol, 2019-01-07;12(2):545-554.
    Species: Mouse
    Sample Types: Cell Culture Supernates
  10. Serum from patients with chronic obstructive pulmonary disease induces senescence-related phenotype in bronchial epithelial cells
    Authors: B Ku?nar-Kam, J Miku?a-Pie, A Witucka, A Romaniuk, N Konieczna, B Rubi?, K Ksi??ek, A Tykarski, H Batura-Gab
    Sci Rep, 2018-08-28;8(1):12940.
    Species: Human
    Sample Types: Cell Culture Supernates
  11. The TRAIL-Induced Cancer Secretome Promotes a Tumor-Supportive Immune Microenvironment via CCR2
    Authors: T Hartwig, A Montinaro, S von Karste, A Sevko, S Surinova, A Chakravart, L Taraborrel, P Draber, E Lafont, F Arce Varga, MA El-Bahrawy, SA Quezada, H Walczak
    Mol. Cell, 2017-02-16;65(4):730-742.e5.
    Species: Human
    Sample Types: Cell Culture Supernates
  12. Pulmonary Epithelial TLR4 Activation Leads to Lung Injury in Neonatal Necrotizing Enterocolitis
    Authors: Hongpeng Jia
    J Immunol, 2016-06-15;0(0):.
    Species: Human
    Sample Types: Cell Culture Supernates
  13. IL-27 inhibits epithelial-mesenchymal transition and angiogenic factor production in a STAT1-dominant pathway in human non-small cell lung cancer.
    Authors: Kachroo P, Lee M, Zhang L, Baratelli F, Lee G, Srivastava M, Wang G, Walser T, Krysan K, Sharma S, Dubinett S, Lee J
    J Exp Clin Cancer Res, 2013-11-25;32(0):97.
    Species: Human
    Sample Types: Cell Culture Supernates
  14. IL-17A and TNF-alpha Exert Synergistic Effects on Expression of CXCL5 by Alveolar Type II Cells In Vivo and In Vitro.
    Authors: Liu Y, Mei J, Gonzales L, Yang G, Dai N, Wang P, Zhang P, Favara M, Malcolm KC, Guttentag S, Worthen GS
    J. Immunol., 2011-01-31;186(5):3197-205.
    Species: Human
    Sample Types: Cell Culture Supernates
  15. Dietary intervention-induced weight loss decreases macrophage content in adipose tissue of obese women.
    Authors: Kovacikova M, Sengenes C, Kováčová Z, Siklova-Vitkova M, Klimcakova E, Polak J, Rossmeislova L, Bajzova M, Hejnova J, Hnevkovska Z, Bouloumie A, Langin D, Stich V
    Int J Obes (Lond), 2010-06-08;35(0):91-8.
    Species: Human
    Sample Types: Plasma
  16. Genetic variation of the human urinary tract innate immune response and asymptomatic bacteriuria in women.
    Authors: Hawn TR, Scholes D, Wang H, Li SS, Stapleton AE, Janer M, Aderem A, Stamm WE, Zhao LP, Hooton TM
    PLoS ONE, 2009-12-15;4(12):e8300.
    Species: Human
    Sample Types: Urine
  17. The inflammatory microenvironment of the aging prostate facilitates cellular proliferation and hypertrophy.
    Authors: Begley LA, Kasina S, Macdonald J, Macoska JA
    Cytokine, 2008-06-24;43(2):194-9.
    Species: Human
    Sample Types: Cell Culture Supernates
  18. Ligation of the FcR gamma chain-associated human osteoclast-associated receptor enhances the proinflammatory responses of human monocytes and neutrophils.
    Authors: Merck E, Gaillard C, Scuiller M, Scapini P, Cassatella MA, Trinchieri G, Bates EE
    J. Immunol., 2006-03-01;176(5):3149-56.
    Species: Human
    Sample Types: Cell Culture Supernates
  19. Increased ENA-78 in the follicular fluid of patients with endometriosis.
    Authors: Wunder DM, Mueller MD, Birkhauser MH, Bersinger NA
    Acta Obstet Gynecol Scand, 2006-01-01;85(3):336-42.
    Species: Human
    Sample Types: Follicular Fluid


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Human CXCL5/ENA-78 DuoSet ELISA
By Joseph Ambrose on 10/27/2020
Sample Tested: Human cell conditioned medium

When I have run this assay with the generic ELISA protocol from RnD Systems the incubation with color substrate is very short. I reduced the detection and capture antibody concentration by half to have more time to stop the reaction and to save reagents.