Human TNF alpha Assay Kit

TNFα is a chemokine mainly produced by macrophages, T cells and neutrophils. TNFα acts as a chemoattractant for neutrophils, and promotes infiltration and activation at inflammation sites.
TNFα is involved in several cancer types for its ability to promote angiogenesis and cell proliferation, as well as inhibit apoptosis.
Cisbio's Human TNFα kit is a homogeneous, add and read assay to measure TNFα 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) 5 pg/mL & 29 pg/mL
Range 29 – 2,500 pg/mL
Time to result 2h at RT
Calibration NIBSC (12/154) value (IU/mL) = 0,1 x HTRF hTNFα value (pg/mL)
Species Human only
Read scientific paper about MALT1 paracaspase inhibition
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.

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(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 hTNFα analysis.

 

How to use HTRF cytokine assays

How to measure multiple cytokines from 1 sample

Analytical performance

Precision

Intra-assay (n=24)

Sample Mean [TNFα] (pg/mL) CV
1 113 6%
2 344 3%
3 1399 6%
  Mean CV 5%

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

Inter-assay (n=4)

Sample [TNFα] (pg/mL) Mean (delta R) CV
1 78 272 11%
2 312 911 7%
3 1250 2894 6%
    Mean CV 6%

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

Accuracy

Dilutional linearity

Sample Dilution factor [TNFα] expected (pg/mL) [TNFα] detected (pg/mL) Recovery
1 1 - 2336 -
3 782 898 115%
4.3 543 567 104%
10.7 218 252 116%
Mean 112%
2 1 - 449 -
3 150 179 119%
Mean 119%

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

Spike and recovery

Sample [TNFα] added (pg/mL) [TNFα] expected (pg/mL) [TNFα] detected (pg/mL) Recovery
1 1,250 1,555 1,523 98%
2 1,250 1,874 1,892 101%

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

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

THP1 cells plated @ 50 & 100 kcells/well were stimulated for 3h with various amounts of LPS.
16 µL of supernatants were then transferred into a white detection plate to be analyzed by the Human TNFα Assay Kit.

2. Inhibition of TNFα secretion in THP1 cells with JTE607

THP1 cells plated @ 100 kcells/well were incubated with various concentrations of JTE 607 for 18h, then stimulated for 3h with 2µg/mL LPS.
16 µL of supernatants were then transferred into a white detection plate to be analyzed by the Human TNFα Assay Kit.

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

BPMCs plated @ 50, 100,200 and 400 kcells/well were stimulated for 3h 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 TNFα Assay Kit.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Human TNF alpha kit - 500 tests62HTNFAPEG
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Human TNF alpha kit - 10,000 tests62HTNFAPEH
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Human TNF alpha kit - 1 x 96 tests62HTNFAPET
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Companion products

DescriptionCat. noProduct insertMSDS
Human TNF alpha kit - 3 Standards62HTNFACD3
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Human TNF alpha kit - Standard62HTNFACDA
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