The HTRF human IL6 kit is designed for the quantification of IL6 release in cell supernatant.

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  • No-wash No-wash
  • Validated on PBMC Validated on PBMC
  • Under 2h bench time Under 2h bench time

The HTRF human IL6 kit is designed for the quantification of IL6 release in cell supernatant.

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Overview

IL6 is a pro-inflammatory cytokine involved in acute-phase reaction, inflammation, and cancer progression. Secreted by T cells, macrophages, and fibroblasts, it induces B and T cell proliferation. It is being studied in a wide variety of research areas including diabetes, Alzheimer's disease, depression, and several cancers. Along with TGF beta, IL6 is the main promoter of T cell differentiation into TH17, a new component of immuno-oncology.


Assessment of serum samples often requires enhanced sensitivity. In some cases, AlphaLISA assays may have sufficient sensitivity to enable the detection of low levels of analytes in serum or plasma. Check out the Human IL-6 AlphaLISA kit and the Human IL-6 AlphaLISA biotin-free kit ’s analytical performances for more information or learn more about AlphaLISA. When assaying, always follow the recommended protocol and avoid highly haemolyzed samples.

Benefits

  • VALIDATED ON PBMC
  • B CELL ACTIVATION READOUT

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 Cisbio 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.
IL6 kit assay principle

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.

To fully understand how to deal with HTRF data processing and also 4PL 1/y² fitting, please visit this page.

Cisbio also worked with Myassays.com to help you in your data analysis. By clicking on this link, you’ll be able to access a free online software to run your IL6 analysis.

Technical specifications of human IL6 kit

Sample size 16 µL
Final assay volume 20 µL
Kit components Lyophilized standard, frozen detection antibodies, buffers &protocol.
LOD &LOQ (in Diluent) 8 pg/mL &34 pg/mL
Range 34 – 7,500 pg/mL
Time to result 2h at RT
Calibration NIBSC (89/548) value (IU/mL) = 0,1 x HTRF hIL6 value (pg/mL)
Species Human only

Intra and inter assay

Intra-assay (n=24)

Sample Mean [IL6] (pg/mL) CV
1 384 5%
2 2522 3%
3 8594 3%
Mean CV 4%

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

Inter-assay (n=4)

Sample [IL6] (pg/mL) Mean (delta R) CV
1 116.5 400 13%
2 268 1805 10%
3 3261 7834 2%
Mean CV 8%

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

Dilutional linearity

Sample
Dilution factor[IL6] expected (pg/mL)[IL6] detected (pg/mL)Recovery
11-2015-
4.3469489104%
7.4271292108%
10.7188210111%
Mean108%
21-354-
4.38382101%
7.4454894%
Mean97%

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

Spike and recovery

Sample
[IL6] added (pg/mL)[IL6] expected (pg/mL)[IL6] detected (pg/mL)Recovery
1325040984103100%

​Recombinant cytokine was added to a serum sample, and the response obtained from a standard curve was compared to the calculated expected values. The 100% of recovery observed validates the sample matrix used for this assay.

Inhibition of IL6 secretion in THP1 cells with JTE607

THP1 cells plated at 100 kcells/well were incubated for 18 h with increasing concentrations of JTE 607 in presence of 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 IL6 Assay.

Inhibition of IL6 secretion in THP1 cells with JTE607.

IL6 secretion in PBMCs stimulated with LPS

PBMC were plated at 50, 100 and 400 kcells/well and stimulated for 18 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 IL6 Assay.

IL6 secretion in PBMCs stimulated with LPS.
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