Human IL6 Assay Kit

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, depression and several cancers.
Along with TGFb, IL6 is the main promoter of T cell differentiation into TH17, a new component of immuno-oncology.
Cisbio's Human IL6 kit is a homogeneous, add and read assay to measure IL6 secretion in supernatant. It offers a fast, efficient alternative to ELISA.

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
Download Insider tips for PBMC isolation Technical Note
Download cytokine release from fresh blood samples application notes

Assay principle

The assay protocol, using a Cisbio 96-well low volume or a 384-well small volume white plate (20 µL final), is described below. Cell supernatant, sample or standard are dispensed directly into the assay plate for the detection by HTRF® reagents. 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 in 96 to 1536-well plates by simply resizing each addition volume proportionally.

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.

Add

Cytokines assay principle add

Read

Cytokines assay principle read

(1) Assay data analysis:

  • "Guidelines for data reduction" button will show you how dealing with HTRF data processing and 4PL 1/y2 fitting.
  • "MyAssays" button will direct you to a free online software able to run IL6 analysis.

 

How to use HTRF cytokine assays

How to measure multiple cytokines from 1 sample

Analytical performance

Precision

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.

Accuracy

Dilutional linearity

Sample Dilution factor [IL6] expected (pg/mL) [IL6] 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 - 354 -
4.3 83 82 101%
7.4 45 48 94%
Mean 97%

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
1 3250 4098 4103 100%

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.

Assay validation

1. IL6 secretion in THP1 cells stimulated with LPS: cell densities

THP1 cells plated @ 50 and 100 kcells/well were stimulated for 18h with LPS.
16 µL of supernatants were then transferred into a white detection plate to be analyzed by the Human IL6 Assay Kit.

2. Inhibition of IL6 secretion in THP1 cells with JTE607

THP1 cells plated @ 100 kcells/well were incubated for 18h with various concentrations of JTE 607 in presence of 2µg/mL LPS.
16 µL of supernatants were then transferred into a white detection plate to be analyzed by the Human IL6 Assay Kit.

3. IL6 secretion in PBMCs stimulated with LPS: cell densities

BPMCs plated @ 50, 100 and 400 kcells/well were stimulated for 18h with various amounts of LPS ( 0, 0.02, 0.2, 2 µg/mL).
16 µL of supernatants were then transferred into a white detection plate to be analyzed by the Human IL6 Assay Kit.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Human IL6 kit - 500 tests62HIL06PEG
Human IL6 kit - 10,000 tests62HIL06PEH
-
Human IL6 kit - 1 x 96 tests62HIL06PET
-

Companion products

DescriptionCat. noProduct insertMSDS
Human IL6 kit - 3 Standards62HIL06CD3
-
-
Human IL6 kit - Standard62HIL06CDA
-

Documents