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QIA68 IL-8 Accucyte® EIA Kit, Human

IL-8 Accucyte® EIA Kit, Human MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms: Interleukin-8, EIA Kit

QIA68
  
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      Overview

      Replacement Information
      Description
      OverviewMeasures IL-8 in serum, plasma, serum-free biological samples, or tissue culture media.
      Catalogue NumberQIA68
      Brand Family Calbiochem®
      SynonymsInterleukin-8, EIA Kit
      Materials Required but Not Delivered• 2-20 µl, 20-200 µl and 200-1000 µ precision pipetters with disposable tips. • Repeating pipettes or multichannel dispenser for washing. • 12 X 75 test tubes. • 25 cc centrifuge tubes. • Deionized H2O. • Graduated serological pipets, 25 ml and/or 10 ml. • Mechanical vortex. • Spectrophotometer capable of measuring absorbance in 96 well plates at a wavelength of 492 nm. • Disposable paper towels. • One 1-liter container.
      References
      Product Information
      Detection methodColorimetric
      DeclarationCalbiochem® is a registered trademark of EMD Chemicals, Inc. Accucyte® is a registered trademark of Cytimmune Sciences, Inc. Interactive Pathways™ is a trademark of EMD Chemicals, Inc.
      Form96 Tests
      Format96-well plate
      Kit containsCoated 96-Well Plate, IL-8 Standard, IL-8 Antibody, IL-8 Conjugate, Streptavidin-Alkaline Phosphatase, Stop Solution, Diluent 1, Diluent 2, Wash Buffer, Color Reagent A, Color Reagent B, Plate Sealers, and a user protocol.
      Applications
      Biological Information
      Assay range0.098 - 100.0 ng/ml
      Assay time5 h
      Sample TypeSerum, plasma, serum-free biological samples, tissue culture media
      Physicochemical Information
      Sensitivity0.098 ng/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 36/37/38

      Irritating to eyes, respiratory system and skin.
      S PhraseS: 26-36

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing.
      Product Usage Statements
      Intended useThe Calbiochem® Accucyte® Human IL-8 is a competitive enzyme immunoassay that measures natural and recombinant forms of the cytokine human Interleukin-8 (IL-8). This assay is for research use only and not for use in diagnostic or therapeutic procedures.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Do not freeze Yes
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCoated 96-Well Plate, IL-8 Standard, IL-8 Antibody, IL-8 Conjugate, Streptavidin-Alkaline Phosphatase, Stop Solution, Diluent 1, Diluent 2, Wash Buffer, Color Reagent A, Color Reagent B, Plate Sealers, and a user protocol.
      Specifications
      Global Trade ITEM Number
      Catalogue Number GTIN
      QIA68 0

      Documentation

      Brochure

      Title
      Angiogenesis and Tumor Metastasis Brochure and Technical Guide
      Kit SourceBook - 2nd Edition EURO
      User Protocol

      Revision20-May-2008 JSW
      SynonymsInterleukin-8, EIA Kit
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      Intended useThe Calbiochem® Accucyte® Human IL-8 is a competitive enzyme immunoassay that measures natural and recombinant forms of the cytokine human Interleukin-8 (IL-8). This assay is for research use only and not for use in diagnostic or therapeutic procedures.
      BackgroundIL-8 is produced by monocytes, fibroblasts, and keratinocytes in response to LPS, IL-1 or TNFα and by T lymphocytes in response to PHA stimulation. Induces the adhesion of neutrophils to endothelial cells. May be involved in chronic inflammation.
      Principles of the assayThe Calbiochem® Accucyte® assay system utilizes pre-coated goat anti-rabbit antibodies to capture a specific IL-8 complex in each sample. Biotinylated IL-8 conjugate (competitive ligand) and sample or standard form a competition reaction for IL-8 specific antibody binding sites. Therefore, as the concentration of IL-8 in the sample increases, the amount of biotinylated IL-8 captured by the antibody decreases. With the addition of streptavidin conjugated alkaline phosphatase (which binds only to the biotinylated IL-8) followed by the addition of the color reagent solution, the amount of biotinylated IL-8 is detected. This results in an inverse relationship between absorbance (Abs) and concentration: the higher the Abs the less IL-8 in the sample.
      Materials providedStandards should be assayed in duplicate. A standard curve must be performed on the same plate and at the same time as the samples. The Accucyte EIA provides sufficient reagents to run two sets of standard curves. • Human IL-8 Coated Plate (Kit Componet No. JA2154): 1 plate, 96 removable wells, coated with goat anti-rabbit antibody. • IL-8 Standard (Kit Componet No. JA2148): 1 vial, containing lyophilized IL-8 standard. • Plate Sealers (Kit Componet No. ): Plate sealers are provided to cover the plate during incubations. • IL-8 Antibody (Kit Componet No. JA2147): 1 vial, containing lyophilized IL-8 antibody. • IL-8 Conjugate (Kit Componet No. JA2149): 1 vial, containing lyophilized, biotinylated IL-8 conjugate. • Streptavidin-Alkaline Phosphatase (Kit Componet No. JA2152): 1 vial, containing lyophilized Strepavidin Alkaline Phosphatase. • Stop Solution (Kit Componet No. JA2153): 1 bottle, 10 ml, 0.5M sulfuric acid. • Diluent 1 (Kit Componet No. JA2145): 1 bottle, 30 ml • Diluent 2 (Kit Componet No. JA2146): 1 bottle, 30 ml • Wash Buffer (Kit Componet No. JA2144): 1 bottle, 50 ml, 20X Wash Buffer. • Color Reagent A (Kit Componet No. JA2150): 1 bottle, 12 ml • Color Reagent B (Kit Componet No. JA2151): 1 bottle, 12 ml -Store unmixed/unused Color Reagent A and Color Reagent B at 4°C for a max of 6 months -Other diluted reagents may be stored at 4°C for a max of 14 days.
      Materials Required but not provided• 2-20 µl, 20-200 µl and 200-1000 µ precision pipetters with disposable tips. • Repeating pipettes or multichannel dispenser for washing. • 12 X 75 test tubes. • 25 cc centrifuge tubes. • Deionized H2O. • Graduated serological pipets, 25 ml and/or 10 ml. • Mechanical vortex. • Spectrophotometer capable of measuring absorbance in 96 well plates at a wavelength of 492 nm. • Disposable paper towels. • One 1-liter container.
      Precautions and recommendations• Warm kit reagents to room temperature before use (let sit at room temperature ~30 min before use.) • Use only the wells provided with the kit. • Do not mix reagents from different kits. • The buffers and reagents used in this kit contain sodium azide. Sodium azide may react with copper and lead plumbing to form highly explosive metal azides. Upon disposal, flush with large amounts of water to prevent azide build-up. Avoid contact with skin. • Do not mouth pipette or ingest any of the reagents. • Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are handled. • Human samples may be contaminated with infectious agents. Do not ingest, expose to open wounds, or breathe aerosols. Dispose of samples properly. • The human source material supplied has been tested by an FDA approved method and found to be non-reactive for HIV-1/2 Antibody, HCV Antibody, a Serologic Test for Syphilis (STS), and Hepatites B Surface Antigen (HbsAg). • Wear disposable gloves and eye protection when handling Stop Solution (0.5 M sulfuric acid).
      Reagent preparation• Human IL-8 Antibody: 1. Serum/Plasma samples: Reconstitute the lyophilized IL-8 antibody with 3.5 ml of Diluent #1 and vortex. 2. Tissue Culture or Biological Samples other than Serum/Plasma: Reconstitute the lyophilized IL-8 antibody with 3.5 ml of Diluent #2 and vortex. • Human IL-8 Standard: Choose from the following options: 1. Serum/Plasma samples: Use Diluent #1 for dilution of standards. 2. Biological samples other than Serum/Plasma: Use Diluent #2 for dilution of standards. 3. Tissue culture supernatant samples: Use Culture media for dilution of standards. Reconstitute the lyophilized IL-8 Standard with 2000 µl of specified diluent and vortex. This will serve as the undiluted standard which has a concentration of 100 ng/ml. To make serial dilutions of the standard, obtain 7 tubes and label them 100, 25, 6.25, 1.56, 0.39, 0.1, and 0 ng/ml. Add 600 µl of specified diluent (see above) into each tube, except the 100 ng/ml tube (first tube) which gets "undiluted" standard. Remove 800 µl from the undiluted, reconstituted standard vial (100 ng/ml) and add it to the first tube. Remove 200 µl from the first tube (100 ng/ml) and add it to the second tube (25 ng/ml) and mix gently. Repeat this procedure until you reach the sixth tube (0.1 ng/ml). The last tube (0 ng/ml) should contain only 600 µl of specified diluent. • Human IL-8 Conjugate: 1. Serum/Plasma samples: Reconstitute the lyophilized IL-8 Conjugate with 3.5 ml of Diluent #1 and vortex. 2. Tissue Culture or Biological Samples other than Serum/Plasma: Reconstitute the lyophilized IL-8 Conjugate with 3.5 ml of Diluent #2 and vortex. • Wash Buffer - Dilute entire contents of concentrated Wash Buffer to 1 liter with deionized water. Stir to homogeneity. • Streptavidin-Alkaline Phosphatase: 1. Serum/Plasma samples: Reconsitute the lyophilized Streptavidin Alkaline Phosphatase with 6.0 ml of Diluent #1 and vortex. 2. Tissue culture or Biological Samples other than Serum/Plasma: Reconstitute the lyophilized Streptavidin Alkaline Phosphatase with 6.0 ml of Diluent #2 and vortex. • Color Reagents - (Do not mix Color Reagents in advance- mix just prior to use) • Allow Color Reagent A and Color Reagent B to come to room temperature. • ~20 ml of the mixed Color Reagent Solution is needed for an entire plate. Use the table below to determine the appropriate volumes of Color Reagent A and Color Reagent B required when only a portion of the Human IL-8 Coated Plate is being used. • Mix appropriate volumes of each reagent, just prior to use, in a clean 15 ml or 25 ml screw-cap centrifuge tube and vortex. The cycling reaction is temperature sensitive, therefore these reagents must be at room temperature prior to use

      Table 1: Reagents
      Detailed protocolThe Calbiochem® IL-8 Accucyte® EIA is provided with removable strips of wells so the assay can be carried out on separate occasions. Since conditions may vary, a standard curve must be determined each time the assay is performed. Both standards and samples should be assayed in duplicate. Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross contamination of reagents or samples. 1. Remove the appropriate number of wells from the foil pouch. Return any unused wells to the foil pouch containing the desiccant pack. SEAL TIGHTLY and store at 4°C. Let all other kit components sit at room temperature until used. Best results will be obtained using reagents at room temperature. 2. Add 100 µl of standards into their designated wells, in DUPLICATE. 3. Add Samples according to the following: • Serum/Plasma samples: For each individual sample, add 100 µl of sample + 200 µl Diluent #1 + 100 µl of Diluent #2 to 12 X 75 test tube and vortex. Add 100 µl of diluted sample to each designated well. • Tissue Culture Samples: To each of the designated wells, add 100 µl of each undiluted tissue culture sample. • Biological Fluid Samples other than Serum/Plasma: To each of the designated wells, add 50 µl of each undiluted sample + 50 µl of Diluent #2. 4. Add 25 µl of reconstituted IL-8 antibody into each well. Cover plate with plate sealer and incubate for 3 h at room temperature. 5. Carefully remove the plate sealer and add 25 µl of reconstituted IL-8 conjugate into each well; seal plate with plate sealer and incubate at room temperature for 30 min. 6. Gently remove the plate sealer and wash the plate 5 times. A thorough washing of the plate is extremely important to reduce background. We recommend using a multi-channel pipette to fill each well with 250 µl of diluted Wash Buffer. Fluid removal from the wells is best accomplished by inverting the plate over a sink and flicking the fluid out of the wells and then blotting the plate on clean paper towels. Using the multichannel pipet add 250 µl of wash buffer to each well; flick and blot the plate. Repeat this procedure a total of 4 times. Dispense 250 µl of diluted Wash Buffer a fifth time and let plate soak for 10 min. After 10 minute soak, blot and aspirate each well to remove any excess fluid. For users of automatic plate washers: it is important to ensure that the wash apparatus is properly maintained and operating correctly. Tubing and tips can easily become clogged, leading to incomplete washing and inadequate aspiration of wells. The result may be poor precision and an unsuitable standard curve. For best results, we recommend 2 cycles of 5 washes each, with a 10 min soak interval between the 2 cycles. 7. Add 50 µl of the reconstituted Streptavidin-Alkaline Phosphatase into each well. Cover with plate sealer and incubate 30 min at room temperature. 8. Gently remove plate sealer. Wash the plate 5 times using the wash method described above. Soak plate with wash buffer for 10 minutes; wash out plate and aspirate each well. 9. Add 200 µl of the prepared Color Reagent Solution into each well. Reseal the plate and incubate at room temperature for approximately 25 min (see below). Please Note: If plate is not adequately washed, there will be no gradations in color in the standard curve. If this occurs, and there is sufficient Color Reagent Solution remaining, wash out the plate and re-add the Color Reagent Solution as described above in Step 9. 10. The Color Reagent incubation time may vary according to laboratory conditions, therefore we recommend that you read the plate at 492 nm during the 15 min incubation to monitor the speed at which color is generated. Plate Reading Scenarios a) If the O.D. for the "0 Dose" should reach 1.6 before the 15 min has elapsed, dispense 50 µl of Stop Solution at this time into each well IN THE SAME ORDER that the Color Reagent Solution was added. b) If the Abs for the "0 Dose" does not reach 1.6 before the 25 min has elapsed, wait until the Abs does reach 1.6 and then dispense 50 µl of Stop Solution into each well IN THE SAME ORDER that the Color Reagent Solution was added. 11. Read the plate a final time at 492 nm. Evaluation of Results 1. Average the duplicate absorbance values for each standard, including the zero, and all sample values. 2. On semi-log graph paper, plot the mean absorbance values for each of the standards on the (linear scale) Y axis, versus the concentration of each standard (ng/ml) on the (log scale) X axis. The standard curve should have a sigmoid shape that shows an inverse relationship between IL-8 concentrations and the corresponding Abs (absorbances). In other words, the greater the concentration of IL-8 in the sample, the lower the Abs, or less red color. 3. Determine the concentration of unknowns by interpolation from the standard curve. There are a variety of Human IL-8 Coated Plate reader software packages available (Softmax, Molecular Devices Corporation, Menlo Park, CA; KinetiCalc, BioTek Instruments, Inc. Winooski, VT) for analysis of Human IL-8 Coated Plate data, which simplifies this process.
      Assay characteristics and examplesSensitivity: 0.098 ng/ml Dynamic Range: 0.098 ng/ml to 100.0 ng/ml Intraassay Error: +/-7.7% Interassay Error: +/-11.2% (+) Crossreactivity: Primate IL-8. (-) Crossreactivity: WHO standards: Human: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-13, IL-15, EGF, FGFα, FGFβ, GM-CSF, G-CSF, M-CSF, IFNα, IFNγ, Leptin, MCP-1, MCP-2, MCP-3, MIP-1α, MIP-1β, PDGF, Rantes, TGFα, TNFα, TNFβ, VEGF. Murine: IL-1α, IL-1β, IL-3, IL-4, IL-6, IL-7, IL-10, GM-CSF, G-CSF, M-CSF, TNFα. Validation By: Parallelism and Quantitative Recovery Sample Matrix: Serum, plasma, serum-free biological samples, & tissue culture Reagent Stability All of the reagents included with the IL-8 Accucyte® EIA have been tested for stability. Reagents should not be used beyond the stated expiration date. Coated plates should be stored at 4°C in the original foil bag containing a desiccant pack.
      Sensitivity0.098 ng/ml
      Assay Range0.098 - 100.0 ng/ml
      Protocol Summary

      Figure 1: Protocol Summary