Interleukin 8 ELISA Kit (IL8)

Details for Product IL8 ELISA Kit No. ABIN612714, Supplier: Log in to see
  • IL8
  • K60
  • interleukin-8
  • il8
  • si:dkey-151b16.2
  • cxcli2
  • Il8
  • Scyb15
  • lungkine
  • weche
  • CXCL8
  • GCP-1
  • GCP1
  • LECT
  • LUCT
  • NAF
  • NAP-1
  • NAP1
  • IL-8
  • AMCF-I
  • EMF-1
  • interleukin 8-like 1
  • interleukin 8
  • chemokine (C-X-C motif) ligand 1-like
  • chemokine (C-X-C motif) ligand 15
  • interleukin 8-like 2
  • interleukin-8-like
  • IL8L1
  • il8
  • IL8
  • LOC422654
  • Cxcl15
  • Il8
  • IL8L2
  • IL*08-02
Dog (Canine) Interleukin 8 ELISA Kit
Kits with alternative reactivity to:
Method Type
Sandwich ELISA
Minimum Detection Limit
1 pg/mL
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Supplier Product No.
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Purpose The AssayMax Human IL-8 ELISA kit is designed for detection of IL-8 in human plasma, tissue extracts or cell culture supernatants
Brand AssayMax
Sample Type Plasma, Cell Culture Supernatant
Analytical Method Quantitative
Detection Method Colorimetric
Specificity This assay recognizes both natural and recombinant human IL-8.
Components IL-8 Microplate: A 96 well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against IL-8. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. IL-8 Standard: Human IL-8 in a buffered protein base (4 ng, lyophilized). Biotinylated IL-8 Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against IL-8 (80µl). 1 MIx Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90µl). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).
Material not included Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water.
Alternative Name Interleukin-8 (IL-8) (IL8 ELISA Kit Abstract)
Background CxCL8 or Interleukin-8 (IL-8) is a member of the CxC chemokine subfamily of cytokines. This basic heparin-binding protein precursor contains 99 amino acids and the mature functional protein comprises 72 amino acids. IL-8 is proinflammatory and primarily mediates the activation and migration of neutrophils from peripheral blood into the sites of inflammation, injury, or infection in the tissue. IL- 8 interacts with two receptors, CxCR1 and CxCR2, to activate leukocytes. Upon activation, both receptors couple to G protein to mediate phosphoinositide-hydrolysis, intracellular Ca2+ mobilization, chemotaxis, and exocytosis. CxCR1 is specific for IL-8 and activates phospholipase D and mediates respiratory burst. IL-8 is involved in a wide variety of physiological and pathological processes, including host defense against bacterial infection, bronchiolitis, arteriosclerosis, autoimmune disorders of skin, bones, and joints, and angiogenesis-dependent disorders such as rheumatoid arthritis, tumor growth, and wound repair.
Research Area Chemokines, Inflammation, Cancer, Cytokines, Receptors, Immunology, Virology
Pathways Cellular Response to Molecule of Bacterial Origin, Regulation of G-Protein Coupled Receptor Protein Signaling, ER-Nucleus Signaling, Hepatitis C, Autophagy
Sample Volume 50 μL
Assay Time < 5 h
Plate Pre-coated
Protocol This assay employs a quantitative sandwich enzyme immunoassay technique that measures IL-8 in less than 5 hours. A polyconal antibody specific for human IL-8 has been pre- coated onto a microplate. IL-8 in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for human IL-8, which is recognized by a streptavidin- peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.
Reagent Preparation

Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. MIx Diluent Concentrate (10x): Dilute the MIx Diluent Concentrate 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 4 ng of human IL-8 Standard with 4 ml of MIx Diluent to generate a standard solution of 1 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare triplicate standard points by serially diluting the IL-8 standard solution twofold with equal volume of MIx Diluent to produce 0.5, 0.25, 0.125, 0.063, 0.031, and 0.016 ng/ml. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [IL-8] (ng/ml) P1 Standard (1 ng/ml) 1.000 P2 1 part P1 + 1 part MIx Diluent 0.500 P3 1 part P2 + 1 part MIx Diluent 0.250 P4 1 part P3 + 1 part MIx Diluent 0.125 P5 1 part P4 + 1 part MIx Diluent 0.063 P6 1 part P5 + 1 part MIx Diluent 0.031 P7 1 part P6 + 1 part MIx Diluent 0.016 P8 MIx Diluent 0.000 Biotinylated IL-8 Antibody (100x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C.

Sample Collection Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles. (EDTA or Heparin can also be used as anticoagulant.) Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Remove serum and assay. Store serum at -20°C or below. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze- thaw cycles. Tissue: Extract tissue samples with 0.1M Tris-buffered saline (pH7.4) containing 0.5% Triton x- 100 and centrifuge at 14000 x g for 30 min. Collect the supernatant and measure the protein concentration. Dilute the tissue extract 1:2 into MIx Diluent and assay. Freeze the remaining extract at -20°C or below. Avoid repeated freeze-thaw cycles.
Assay Procedure

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of Standard or sample per well. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer. Invert the plate and decant the contents, and hit it 4-5 times on absorbent paper towel to complete remove liquid at each step. Add 50 µL of Biotinylated IL-8 Antibody to each well and incubate for two hours. Wash five times with 200 µL of Wash Buffer as above. Add 50 µL of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash five times with 200 µL of Wash Buffer as above. Add 50 µL of Chromogen Substrate per well and incubate for approximately 7 minutes or till the optimal blue color density develops. Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately.

Calculation of Results

Calculate the mean value of the duplicate or triplicate readings for each standard and sample. To generate a Standard Curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit. Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

Assay Precision Intra-assay and inter-assay coefficients of variation were 5.0% and 7.2% respectively.
Restrictions For Research Use only
Handling Advice The kit should not be used beyond the expiration date.
Storage 4 °C/-20 °C
Storage Comment Store kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened MIx Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along with zip-seal. May be stored for up to 1 month in a vacuum desiccator.
Supplier Images
ELISA image for Interleukin 8 (IL8) ELISA Kit (ABIN612714) Interleukin 8 (IL8) ELISA Kit
Product cited in: Ahmed, Hamed, Omar: "Clinical utility of certain biomarkers as predictors of breast cancer with or without metastasis among Egyptian females." in: Tumour biology, Vol. 36, Issue 2, pp. 815-22, 2015 (PubMed).

Twu, Dessí, Vu, Mercer, Stevens, de Miguel, Rappelli, Cocco, Clubb, Fiori, Johnson: "Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth, invasiveness, and inflammatory responses." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 111, Issue 22, pp. 8179-84, 2014 (PubMed).

Twu, de Miguel, Lustig, Stevens, Vashisht, Wohlschlegel, Johnson: "Trichomonas vaginalis Exosomes Deliver Cargo to Host Cells and Mediate Host∶Parasite Interactions." in: PLoS pathogens, Vol. 9, Issue 7, pp. e1003482, 2013 (PubMed).

Background publications Harada, Mukaida, Matsushima: "Interleukin 8 as a novel target for intervention therapy in acute inflammatory diseases." in: Molecular medicine today, Vol. 2, Issue 11, pp. 482-9, 1997 (PubMed).

Jones, Wolf, Qin, Mackay, Baggiolini: "Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 93, Issue 13, pp. 6682-6, 1996 (PubMed).

LHeureux, Bourgoin, Jean, McColl, Naccache: "Diverging signal transduction pathways activated by interleukin-8 and related chemokines in human neutrophils: interleukin-8, but not NAP-2 or GRO alpha, stimulates phospholipase D activity." in: Blood, Vol. 85, Issue 2, pp. 522-31, 1995 (PubMed).

Watson, Camera-Benson, Palmer-Crocker, Pober: "Variability among human umbilical vein endothelial cultures." in: Science (New York, N.Y.), Vol. 268, Issue 5209, pp. 447-8, 1995 (PubMed).

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