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 & disposal requirements under European REACH regulations (EC 1907/2006).
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.
Assay Points |
10000
|
---|---|
Assay Target Type |
Kit
|
Assay Technology |
HTRF
|
Brand |
HTRF
|
Quantity |
1
|
Shipping Conditions |
Shipped in Dry Ice
|
Therapeutic Area |
Metabolism/Diabetes
NASH/Fibrosis
Oncology & Inflammation
|
Unit Size |
10,000 Assay Points
|
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.
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.
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-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.
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% |
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% |
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.
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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