Human IFN gamma Assay Kit

IFNγ is a pro-inflammatory cytokine secreted by T cells and NK cells in response to viral infection and other inflammatory conditions. It induces the expression of class II MHC, macrophage activation, and the Immunoglobulin switch in B cells. IFNγ plays a major role in innate and adaptive immunity by promoting T cell differentiation into TH1 and the development of Treg, but also by inhibiting T cell differentiation into TH17. Its anti-tumor properties have been used in the treatment of osteoporosis and it is of great interest in immuno-oncology where it improves survival in patients with bladder or ovarian carcinoma.
Cisbio's Human IFNγ kit is a homogeneous, add and read assay to measure IFNγ 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) 14 pg/mL & 21 pg/mL
Range 21 – 4,000 pg/mL
Time to result Overnight at RT
Calibration NIBSC (82/587) value (IU/mL) = 0,019 x HTRF hIFNγ 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 IFNγ analysis.

 

How to use HTRF cytokine assays

How to measure multiple cytokines from 1 sample

Analytical performance

Precision

Intra-assay (n=24)

Sample Mean [IFNγ] (pg/mL) CV
1 209 9%
2 835 11%
3 4367 7%
  Mean CV 9%

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

Inter-assay (n=4)

Sample [IFNγ] (pg/mL) Mean (delta R) CV
1 98 178 14%
2 432 788 9%
3 1905 3170 6%
    Mean CV 9.7%

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

Accuracy

Dilutional linearity

Sample Dilution factor [IFNγ] expected (pg/mL) [IFNγ] detected (pg/mL) Recovery
1 1 - 150 -
2.1 72.6 66 91%
4.3 35 30 86%
Mean 88%
2 1 - 1953 -
2.1 942 959 102%
4.3 454 450 99%
Mean 100%

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

Spike and recovery

Sample [IFNγ] added (pg/mL) [IFNγ] expected (pg/mL) [IFNγ] detected (pg/mL) Recovery
1 2,000 2,421 2,086 86%
2 2,000 3,360 3,363 100%

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 observed validates the sample matrix used for this assay.

Assay validation

IFNγ stimulation in PBMCs stimulated with PMA and Ionomycin: cell densities

BPMCs plated @ 50, 100,200 and 400 kcells/well were stimulated for 3h with various amounts of PMA (0, 1, 50 ng/mL) added to a 500 ng/mL Ionomycin solution. 16 µL of supernatants were then transferred into a white detection plate to be analyzed by the Human IFNγ Assay Kit.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Human IFN gamma kit - 500 tests62HIFNGPEG
Human IFN gamma kit - 10,000 tests62HIFNGPEH
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Human IFN gamma kit - 1 x 96 tests62HIFNGPET
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Companion products

DescriptionCat. noProduct insertMSDS
Human IFN gamma kit - Standard62HIFNGCDA
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