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HTRF HP Human TNFα Detection Kit, 96 assay points

TNF alpha Product image
HTRF HP Human TNFα Detection Kit, 96 assay points
TNF alpha Product image
TNF alpha Product image
TNF alpha Product image
TNF alpha Product image
TNF alpha Product image

The HTRF HP Human TNF-α kit is designed for the simple and rapid quantification of TNF-α proteins in cell supernatants, offering a no-wash alternative to ELISA. This kit is a new and improved version of the existing kit, 62HTNFAPEG.

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Feature Specification
Application Protein Quantification
Dynamic Range 37 – 5 500 pg/mL
Limit of Detection 9 pg/mL
Limit of Quantification 16 pg/mL
Sample Volume 16 µL
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The HTRF HP Human TNF-α kit is designed for the simple and rapid quantification of TNF-α proteins in cell supernatants, offering a no-wash alternative to ELISA. This kit is a new and improved version of the existing kit, 62HTNFAPEG.

View product information
Click to copy promo code to clipboard.
img-icon-10-off-white-yellow.svg

SAVE 10% on your first Revvity.com order. Use promo code below.

HELLO10

Terms and conditions apply.

Product Variants
Unit Size: 500 assay points
Part #:
62HTNFAV2PEG
List Price
USD 1,103.00
Your online price:
Unit Size: 10,000 assay points
Part #:
62HTNFAV2PEH
List Price
USD 10,290.00
Your online price:
Unit Size: 96 assay points
Part #:
62HTNFAV2PET
List Price
USD 652.00
Your online price:
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).
Total list price:
USD 652.00
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TNF alpha Product image
TNF alpha Product image
HTRF HP Human TNFα Detection Kit, 96 assay points
TNF alpha Product image
TNF alpha Product image
TNF alpha Product image
HTRF HP Human TNFα Detection Kit, 96 assay points
TNF alpha Product image
TNF alpha Product image

Product information

Overview

TNF alpha is a cytokine mainly produced by macrophages, T cells, and neutrophils. TNF alpha acts as a chemoattractant and promotes infiltration and activation at inflammation sites. TNF alpha is involved in several cancer types due to its ability to promote angiogenesis and cell proliferation, as well as to inhibit apoptosis.

Specifications

Application
Protein Quantification
Brand
HTRF
Detection Modality
HTRF
Dynamic Range
37 – 5 500 pg/mL
Limit of Detection
9 pg/mL
Limit of Quantification
16 pg/mL
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target
TNFα
Target Class
Cytokines
Target Species
Human
Technology
TR-FRET
Therapeutic Area
Inflammation
Unit Size
96 assay points

How it works

Principle of the HTRF HP human TNF alpha assay

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.

Assay protocol TNFalpha how it works 62HTNFAV2PEG 62HTNFAV2PEH 62HTNFAV2PET
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.

Assay details

HTRF HP human TNF alpha standard curve

The HP human TNF alpha standard curve is generated in the assay Diluent 1, provided in the kit.

assay details standard curve TNF alpha 62HTNFAV2PEG 62HTNFAV2PEH 62HTNFAV2PET.jpg

 

Human TNF alpha assay specifications
Sample size 16 µL
Final assay volume 20 µL
Kit components Lyophilized standard, frozen detection antibodies, buffers & protocol
LOD & LOQ (in Diluent) 9 pg/mL & 16 pg/mL
LOD & LOQ (in DMEM+10%FCS) 14 & 22 pg/mL
LOD & LOQ (in RPMI+10%FCS) 17 & 23 pg/mL
Range 37 – 5 500 pg/mL
Time to result 2h at RT
Correspondance to I.S. NIBSC (12/154) value (IU/mL) = 0.1 x HTRF HP hTNFα value (pg/mL)
Species Human only

 

Analytical performance

Intra-assay precision table

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

Sample Mean [TNFα](pg/mL) CV
1 180 5%
2 390 4%
3 2 216 2%
  Mean CV 4%

 

Inter-assay precision table

Each of the samples was measured in 3 independent experiments (3 days), and the % CV was calculated for each sample.

Sample Mean [TNFα](pg/mL) CV
1 92 9%
2 513 7%
3 2 424 4%
  Mean CV 7%

 

Dilutional linearity

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

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

1

 1 1258 1258.01 100%
2 629 646.61 103%
4 315 324.18 103%
8 157 180.65 115%
16 79.0 83.88 106%

Mean

 105%

2

 1 628.4 628.41 100%
1.5 419 439.20 105%
2.2 279 270.06 97%
3.4 186 182.35 98%
5 124 110.16 89%

Mean

 98%

 

Spike and recovery

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 validated the sample matrix used for this assay.

Sample

[TNFα] Spiked Sample

(pg/mL)

[TNFα] Standard

(pg/mL)

Expected

(pg/mL)

Measured

(pg/mL)

 Recovery

1

 125 98 223 221 99%
1196 1321 1305 99%

2

 154 98 252 269 107%
1196 1350 1360 101%

 

Mean CV

 101%

 

Cross reactivities

Cross reactivities were assessed using recombinant proteins from the TNF alpha cytokine family. Proteins were tested up to 5,500 pg/mL, and standard curves were generated for each. Signals were interpolated on the assay standard curve to check concentrations.

Tested Protein Cross Reactivity
Human TNF alpha 100%
Mouse TNF alpha 0%
Bovine TNF alpha 0%

 

 

Assay validation

Human TNF alpha secretion in PBMC cells stimulated with LPS

PBMCs plated at 50, 100, 200, and 400 kcells/well were stimulated for 3h with LPS at 0.2 µg/mL. Then 16 µL of supernatants were transferred into a white detection plate (384 low volume) to be analyzed using the HTRF HP Human TNF alpha assay Kit.

TNF alpha 62HTNFAV2PEG 62HTNFAV2PEH 62HTNFAV2PET

 

Effect of JTE-607 on THP-1 cell induced TNF alpha release

THP-1 cells plated at 100 kcells/well were stimulated for 18h with increasing concentrations of the inhibitor JTE 607 in presence of 2 µg/mL LPS. Then 16 µL of supernatants were transferred into a white detection plate (384 low volume) to be analyzed using HTRF HP Human TNF alpha assay kit.

assay validation JTE 607 THP1TNF alpha 62HTNFAV2PEG 62HTNFAV2PEH 62HTNFAV2PET.jpg

 

Effect of JTE-607 on PBMC induced TNF alpha release

PBMCs plated at 200 and 400 kcells/well were stimulated for 18h with increasing concentrations of the inhibitor JTE-607 in presence of 2 µg/mL LPS. Then 16 µL of supernatants were transferred into a white detection plate (384 low volume) to be analyzed using the HTRF HP Human TNF alpha assay kit.

assay validation JTE 607 PBMC TNF alpha 62HTNFAV2PEG 62HTNFAV2PEH 62HTNFAV2PET.jpg

 

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