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HTRF Human TNα Detection Kit, 10,000 Assay Points

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

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).

Feature Specification
Application Protein Quantification
Sample Volume 16 µL

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

Product Variants
Unit Size: 96 Assay Points
Part #:
62HTNFAPET
List Price
USD 652.48
Unit Size: 500 Assay Points
Part #:
62HTNFAPEG
List Price
USD 1,103.00
Unit Size: 10,000 Assay Points
Part #:
62HTNFAPEH
List Price
USD 10,290.00

Overview

TNF alpha is a cytokine mainly produced by macrophages, T cells, and neutrophils. TNF alpha acts as a chemoattractant neutrophils, and promotes infiltration and activation at inflammation sites. TNF alpha is involved in several cancer types 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 detection of low levels of analytes in serum or plasma. When assaying, always follow recommended protocol and avoid highly haemolyzed samples.

Specifications

Application
Protein Quantification
Brand
HTRF
Detection Modality
HTRF
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target Class
Cytokines
Target Species
Human
Technology
TR-FRET
Therapeutic Area
Metabolism/Diabetes
NASH/Fibrosis
Oncology & Inflammation
Unit Size
10,000 Assay Points

Video gallery

How it works

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 Revvity 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.

 

1cytokines-how-it-works-assay-principle-human-il13-62hil13peg-62hil13peh.svg

 

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,  Revvity also worked with Myassays.com to help you in your data analysis. you’ll be able to access a free online software to run your TNF alpha analysis.

 

Assay details

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

 

 

Analytical performance

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%

 

 

Assay validation

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.

 

1assay-validation-human-tnf-alpha-2.svg

 

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.

 

2assay-validation-human-tnf-alpha-3.svg

 

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