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HTRF Human IL-8 Detection Kit, 96 Assay Points

HTRF human IL8 kit is designed quantification of human IL8 release in cell supernatant.

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).
Part Number: 62HIL08PET
Unit Size: 96 Assay Points
Part Number: 62HIL08PEG
Unit Size: 500 Assay Points
Part Number: 62HIL08PEH
Unit Size: 10,000 Assay Points
  • Also called CXCL8, IL8 is a chemokine mainly produced by macrophages, T cells, and neutrophils. IL8 acts as a chemoattractant neutrophils, and promotes infiltration and activation at inflammation sites. IL8 is involved in several cancer types due to its ability to promote angiogenesis and cell proliferation, as well as to inhibit apoptosis.

    Assessment of serum samples often requires enhanced sensitivity. In some cases, AlphaLISA assays may have sufficient sensitivity to enable detection of low levels of analytes in serum or plasma.

  • Assay Points
    96
    Assay Target Type
    Kit
    Assay Technology
    HTRF
    Brand
    HTRF
    Quantity
    1
    Therapeutic Area
    Oncology & Inflammation
    Unit Size
    96 Assay Points
  • Assay principle

    Cell supernatant, sample, or standard is dispensed directly into the assay plate for the detection by HTRF® reagents (384-well low-volume white plate or Revvity low-volume 96-well plate in 20 µl). The antibodies labeled with the HTRF donor and acceptor are pre-mixed and added in a single dispensing step, to further streamline the assay procedure. The assay can be run up to a 1536-well format by simply resizing each addition volume proportionally.

    cytokines-how-it-works-assay-principle-human
    Assay data analysis

    The 4 Parameter Logistic (4PL) curve is commonly recommended for fitting an ELISA standard curve. This regression enables the accurate measurement of an unknown sample across a wider range of concentrations than linear analysis, making it ideally suited to the analysis of biological systems like cytokine releases.

    Revvity also worked with Myassays.com to help you in your data analysis.

     

  • Technical specifications of human IL8 kit
    Sample size 16 µL
    Final assay volume 20 µL
    Kit components Lyophilized standard, frozen detection antibodies, buffers &protocol.
    LOD &LOQ (in Diluent) 6 pg/mL &32 pg/mL
    Range 32 4,000 pg/mL
    Time to result 1h at RT
    Calibration NIBSC (89/520) value (IU/mL) = 0,01 x HTRF hIL8 value (pg/mL)
    Species Human only

     

  • Intra and inter assay

    Intra-assay (n=24)

    Sample Mean [IL8] (pg/mL) CV
    1 58 9%
    2 314 4%
    3 2074 2%
      Mean CV 5%


    Each of the 3 samples was measured 24 times, and % CV was calculated for each sample.

    Inter-assay (n=4)

    Sample [IL8] (pg/mL) Mean (delta R) CV
    1 78 273 2%
    2 376 1504 12%
    3 1818 5990 5%
        Mean CV 6.3%


    Each of the samples was measured in 4 different experiments, and % CV was calculated for each sample.

    Dilutional linearity
    Sample Dilution Factor [IL8] expected (pg/mL) [IL8] detected (pg/mL) Recovery
    1 1 - 2015 -
    4.3 469 489 104%
    7.4 271 292 108%
    10.7 188 210 111%
    Mean -   108%
    2 1   399 -
    4.3 93 85 91%
    7.4 54 56 103%
    Mean     97%


    The excellent % of recovery obtained from these experiments show the good linearity of the assay.


    Spike and recovery
    Sample [IL8] added (pg/mL) [IL8] expected (pg/mL) [IL8] detected (pg/mL) Recovery
    1 5000 5179 5008 97%
    2 5000 6047 5682 94%


    ​The same amount of recombinant cytokine was added to 2 different serum samples, and the set of responses obtained from a standard curve was compared to the calculated expected values. The ~ 100% of recovery found validates the sample matrix used for this assay.

     

  • Inhibition of IL8 secretion in THP1 cells with JTE607

    THP1 cells plated at 100 k cells/well were incubated with increasing concentrations of JTE 607 for 18h, then stimulated for 3 h with 2 µg/mL LPS. 16 µL of supernatants were then transferred into a white detection plate (384 low volume) to be analyzed by the Human IL8 Assay.

    assay-validation-human-il8-2
    IL8 secretion in PBMCs stimulated with LPS

    PBMC plated at 50, 100, 200, and 400 k cells/well were stimulated for 3 h with increasing concentrations of LPS (0, 0.02, 0.2, 2 µg/mL). 16 µL of supernatants were then transferred into a white detection plate (384 low volume) to be analyzed by the Human IL8 Assay.

    assay-validation-human-il8

     

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