Human TNF alpha ELISA Kit (ab181421)
Key features and details
- One-wash 90 minute protocol
- Sensitivity: 4.32 pg/ml
- Range: 15.63 pg/ml - 1000 pg/ml
- Sample type: Cell culture supernatant, Cit plasma, EDTA Plasma, Hep Plasma, Serum
- Detection method: Colorimetric
- Assay type: Sandwich (quantitative)
- Reacts with: Human
Overview
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Product name
Human TNF alpha ELISA Kit
See all TNF alpha kits -
Detection method
Colorimetric -
Precision
Intra-assay Sample n Mean SD CV% PBMC media 8 2.5% Inter-assay Sample n Mean SD CV% PBMC media 3 3.1% -
Sample type
Cell culture supernatant, Serum, Hep Plasma, EDTA Plasma, Cit plasma -
Assay type
Sandwich (quantitative) -
Sensitivity
4.32 pg/ml -
Range
15.63 pg/ml - 1000 pg/ml -
Recovery
Sample specific recovery Sample type Average % Range Cell culture supernatant 110 105% - 113% Serum 88 83% - 93% Hep Plasma 98 89% - 110% EDTA Plasma 100 95% - 104% Cit plasma 91 86% - 97% -
Assay time
1h 30m -
Assay duration
One step assay -
Species reactivity
Reacts with: Human -
Product overview
Human TNF alpha ELISA kit (ab181421) is a single-wash 90 min sandwich ELISA designed for the quantitative measurement of TNF alpha protein in human serum, plasma and culture media. It uses our proprietary SimpleStep ELISA® technology. Quantitate human TNFa with 4.32 pg/mL sensitivity.
SimpleStep ELISA® technology employs capture antibodies conjugated to an affinity tag that is recognized by the monoclonal antibody used to coat our SimpleStep ELISA® plates. This approach to sandwich ELISA allows the formation of the antibody-analyte sandwich complex in a single step, significantly reducing assay time. See the SimpleStep ELISA® protocol summary in the image section for further details. Our SimpleStep ELISA® technology provides several benefits:
-Single-wash protocol reduces assay time to 90 minutes or less
-High sensitivity, specificity and reproducibility from superior antibodies
-Fully validated in biological samples
-96-wells plate breakable into 12 x 8 wells stripsA 384-well SimpleStep ELISA® microplate (ab203359) is available to use as an alternative to the 96-well microplate provided with SimpeStep ELISA® kits.
ASSAY SPECIFICITY This kit recognizes both native and recombinant human TNF alpha protein in serum, plasma, and cell culture supernatant samples only.
CROSS REACTIVITY Recombinant proteins were prepared at 1000 pg/mL and assayed for cross reactivity. No cross reactivity was found for the following targets: - Human IL-2 - Human IL-4 - Human IL-1 alpha - Human IFN gamma - Human TNF beta - Human TNF R1.
INTERFERENCE Recombinant Human TNF R1 was prepared at 1000 pg/mL and tested for interference. No interference with was observed.
SPECIES REACTIVITY This kit recognizes human TNF alpha protein. Other species reactivity was determined by measuring 1000 pg/mL recombinant proteins of various species, interpolating the protein concentrations from the human standard curve, and expressing the interpolated concentrations as a percentage of the protein concentration assayed at the same dilution. Reactive species: Primate
ReactivityCALIBRATION This immunoassay is calibrated against a highly purified human TNF alpha. The NIBSC/WHO unclassified purified human TNF alpha preparation 12/154 was evaluated in this kit. The dose response curve of the unclassified standard TNF alpha parallels the SimpleStep standard curve. To convert sample values obtained with the SimpleStep Human TNF alpha kit to approximate NIBSC 12/154 International units, use the equation below.
NIBSC (12/154) approximate value (IU/mL) = 0.094 x SimpleStep Human TNF alpha value (pg/mL).
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Notes
TNF-alpha, also known as cachectin or TNFSF1A, is the prototypic ligand of the TNF superfamily which plays a central role in inflammation, apoptosis, proliferation, invasion, angiogenesis, metastasis and morphogenesis. It is expressed on macrophages, endothelial, epithelial and tumor cells as a 26kDa transmembrane protein. TNF-alpha is cleaved by proteolytic processing into six chains: (1) TNF membrane form, (2) Intracellular domain 1, (3) Intracellular domain 2, (4) C-domain 1, (5) C-domain 2 and (6) TNF soluble form. Signaling from TNF-alpha differs depending on the type of ligand initiating the signaling event (intracellular, membrane or soluble). As an example, the membrane form of TNF-alpha appears to mediate anti-tumorigenic therapeutic responses whereas the soluble ligand is linked to inflammation and proliferation.
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Platform
Microplate
Properties
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Storage instructions
Store at +4°C. Please refer to protocols. -
Components 1 x 96 tests 10X Wash Buffer PT (ab206977) 1 x 20ml Antibody Diluent 4BR 1 x 6ml Human TNFa Capture Antibody 1 x 600µl Human TNFa Detector Antibody 1 x 600µl Human TNFa Lyophilized Recombinant Protein 2 vials Plate Seals 1 unit Sample Diluent NS (ab193972) 1 x 50ml SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit Stop Solution 1 x 12ml TMB Development Solution 1 x 12ml -
Research areas
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Function
Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation. -
Involvement in disease
Genetic variations in TNF are a cause of susceptibility psoriatic arthritis (PSORAS) [MIM:607507]. PSORAS is an inflammatory, seronegative arthritis associated with psoriasis. It is a heterogeneous disorder ranging from a mild, non-destructive disease to a severe, progressive, erosive arthropathy. Five types of psoriatic arthritis have been defined: asymmetrical oligoarthritis characterized by primary involvement of the small joints of the fingers or toes; asymmetrical arthritis which involves the joints of the extremities; symmetrical polyarthritis characterized by a rheumatoidlike pattern that can involve hands, wrists, ankles, and feet; arthritis mutilans, which is a rare but deforming and destructive condition; arthritis of the sacroiliac joints and spine (psoriatic spondylitis). -
Sequence similarities
Belongs to the tumor necrosis factor family. -
Post-translational
modificationsThe soluble form derives from the membrane form by proteolytic processing.
The membrane form, but not the soluble form, is phosphorylated on serine residues. Dephosphorylation of the membrane form occurs by binding to soluble TNFRSF1A/TNFR1.
O-glycosylated; glycans contain galactose, N-acetylgalactosamine and N-acetylneuraminic acid. -
Cellular localization
Secreted and Cell membrane. - Information by UniProt
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Alternative names
- APC1
- APC1 protein
- Cachectin
see all -
Database links
- Entrez Gene: 7124 Human
- Omim: 191160 Human
- SwissProt: P01375 Human
- Unigene: 241570 Human
Images
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SimpleStep ELISA Protocol Diagram
SimpleStep ELISA technology allows the formation of the antibody-antigen complex in one single step, reducing assay time to 90 minutes. Add samples or standards and antibody mix to wells all at once, incubate, wash, and add your final substrate. See protocol for a detailed step-by-step guide.
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Human TNF alpha standard curve comparisonStandard Curve comparison between human TNF alpha SimpleStep ELISA kit and traditional ELISA kit from leading competitor. SimpleStep ELISA kit shows increased sensitivity.
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Example of human TNF alpha standard curve in Sample Diluent NS.The TNF alpha standard curve was prepared as described in Section 10. Raw data values are shown in the table. Background-subtracted data values (mean +/- SD) are graphed.
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Raw data value for example of TNF standard curve in Sample Diluent NSRaw data values are shown in the table
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Interpolated concentrations of native TNF alpha in human PBMC cell culture supernatant samples.PBMC cells were treated with 1.5% PHA-M for 46 hours in 10F RPMI. The concentrations of TNF alpha were measured in duplicates, interpolated from the TNF alpha standard curve and corrected for sample dilution. Undiluted samples are PHA-M treated PBMC cell culture supernatant 10%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean TNF alpha concentration was determined to be 7326.31 pg/mL in PHA-M treated PBMC cell culture supernatant.
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Interpolated concentrations of native TNF alpha PBMC cell culture supernatant samples.PBMC cells were stimulated with 1.5% PHA-M or vehicle control in 10F RPMI and incubated for 46 hours. The concentrations of TNF alpha were measured in duplicate and interpolated from the TNF alpha standard curves. Undiluted samples are PHA-M stimulated PBMC cell culture supernatant 10% and unstimulated PBMC cell culture supernatant 10%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean TNF alpha concentration was back calculated determined to be 7265.71 pg/mL in PHA-M stimulated PBMC cell culture supernatant and undetectable in the unstimulated PBMC control.
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Linearity of dilution - native TNF alphaNative TNF alpha was measured in biological samples in a 2-fold dilution series. Sample dilutions are made in Sample Diluent NS.
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Linearity of dilution - recombinant TNF alphaRecombinant TNF alpha was spiked biological samples and diluted in a 2-fold dilution series in Sample Diluent NS. Twenty individual healthy human female/male donors were measured in duplicate for the presence of TNF alpha. All values were below the detectable range of the assay
NL – Non-Linear -
Sandwich ELISA - Human TNF alpha ELISA kit (ab181421)Human TNF alpha concentration was interpolated from the standard curve. Supernatants from cell culture samples were serially diluted and assessed by the Human TNF alpha ELISA kit (ab181421). Wild-type THP-1 and TNF alpha knockout THP-1 (ab273761) cells were assessed in duplicate (n=2). Cells were either treated with 100 ng/ml LPS for 16 h to induce expression of TNF alpha or not treated with LPS. Data are represented as the mean and error bars represent standard deviation.”
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Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421)To learn more about the advantages of recombinant antibodies see here.
