Human TNF alpha kit HTRF®

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

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

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



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. 



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.
TNF alpha 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 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)

SampleMean [TNFα] (pg/mL)CV

Mean CV5%

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

Mean CV6%

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.​

Dilution factor[TNFα] expected (pg/mL)[TNFα] detected (pg/mL)Recovery

Spike and recovery

[TNFα] added (pg/mL)[TNFα] expected (pg/mL)[TNFα] detected (pg/mL)Recovery

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.

Inhibition of TNF alpha secretion in THP1 cells with JTE607.

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.
TNF alpha secretion in PBMCs stimulated with LPS.

Celular High Throughput Screening assays using HTRF detection of cytokines

In collaboration with GSK - Scientific Presentations

HTRF Cytokine cell-based assays

Monitor immune regulation the simple way - Flyers

Biomarker and Cell Signaling Assays for Fibrosis and NASH

HTRF and Alpha solutions for NASH - Flyers

Inflammation cell by cell

HTRF solutions for each cell type - Flyers

HTRF assays for Oncology and Inflammation

Signaling in the Immune System - Brochures

HTRF measurement of cytokine release from fresh blood samples

In collaboration with Blood assay solution - Application Notes

Cytokine secretion profile on PBMCs

One sample to measure them all - Posters

Guidelines from PBMC isolation to cytokine assay optimisation

Perform and optimize cytokine assays on PBMC - Technical Notes

How to freeze PBMC

Guideline for PBMC freezing - Technical Notes

Webinar: NASH - Fibrosis Research and Drug Discovery

Featuring a panel of experts - Videos

How to measure multiple cytokines from 1 sample

What to expect at the bench - Videos

Product Insert TNF a (h) Kit / 62HTNFAPEG-62HTNFAPEH

62HTNFAPEG-62HTNFAPEH - Product Insert

HTRF makes cytokine detection easy

A fun video introducing you to cytokines assays with HTRF - Videos

STING HTRF offer to bridge innate and adaptive immunity

cGAS-STING signaling pathway from A to Z - Brochures

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

Cytokine assays: a guide to success with HTRF

Setting up a successful cytokine assay - Guides

Functional response to PD1 blockade rapidly assessed through HTRF IFNγ quantification

For researcher working on checkpoint inhibitors - Application Notes

Portfolio of Life Sciences Cytokine Assays

This leaflet presents the analytical performances of each assay - Brochures

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

An innate and adaptive immunity recap

Insight into the diversity of cells & signaling pathways - Guides

Molecular basis of neuroinflammation and neurodegeneration diseases

The essential guide for extending your knowledge on the molecular mechanisms of neurodegenerative diseases - Guides

Cytokine Portfolio Flyer

Discover no-wash cytokine assay solutions - Flyers

Innovative solutions in Virology research

Kits and reagents for virology research - Flyers

On-demand webinar: Linking Neuroinflammation and Neurodegeneration

New insight into neuroinflammation research - Videos

Neutralizing antibodies in Autoimmunity

Power up your autoimmunity research projects with this application note - Application Notes

Assays for autoimmunity research

Advance your research on autoimmune diseases - Flyers

Advance your research on Fibrosis

Kits and reagents for Fibrosis research - Flyers

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

Let's find your reader