Human TNF alpha kit

The HTRF human TNFα kit is designed for the quantification of human TNF alpha release in cell supernatant.

No-wash
Validated on PBMC
Under 2h bench time

The HTRF human TNFα kit is designed for the quantification of human TNF alpha release in cell supernatant.

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Overview

TNF alpha is a cytokine mainly produced by macrophages, T cells, and neutrophils. TNF alpha acts as a chemoattractant for neutrophils, and promotes infiltration and activation at inflammation sites. TNF alpha is involved in several cancer types for 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 the detection of low levels of analytes in serum or plasma. Check out the Human TNFα AlphaLISA 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
VALIDATED ON THP1 CELLS

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.

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 TNF alpha analysis.

Technical specifications of human TNF alpha kit

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 hTNFa value (pg/mL)
Species	Human only

Intra and inter assay

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	[TNFa] (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.

Dilutional linearity

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

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%

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%

Inhibition of TNF alpha secretion in THP1 cells with JTE607

THP1 cells plated at 100 kcells/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 TNFa Assay.

TNF alpha secretion in PBMCs stimulated with LPS

PBMC plated at 50, 100, 200 and 400 kcells/well were stimulated for 3h 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 TNFa Assay.

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Human TNF alpha kit

Overview

Benefits

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