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  1. Link

    products/elisa/human-tnf-alpha-elisa-kit-ab181421.pdf

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Immunology Innate Immunity Cytokines Necrosis Factors
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Validated using a knockout cell lineRecombinantSimpleStep

Human TNF alpha ELISA Kit (ab181421)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (3) Submit a question References (106)

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SimpleStep ELISA Protocol Diagram
  • Human TNF alpha standard curve comparison
  • Example of human TNF alpha standard curve in Sample Diluent NS.
  • Raw data value for example of TNF standard curve in Sample Diluent NS
  • Interpolated concentrations of native TNF alpha in human PBMC cell culture supernatant samples.
  • Interpolated concentrations of native TNF alpha PBMC cell culture supernatant samples.
  • Linearity of dilution - native TNF alpha
  • Linearity of dilution - recombinant TNF alpha
  • Sandwich ELISA - Human TNF alpha ELISA kit (ab181421)
  • Sandwich ELISA - 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

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Overview

  • 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 strips


    A 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
    Reactivity < 3% was determined for the following species: Mouse/Rat


    CALIBRATION 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).

  • 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.

  • Platform

    Microplate

Properties

  • 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

    • Immunology
    • Innate Immunity
    • Cytokines
    • Necrosis Factors
    • Immunology
    • Innate Immunity
    • Cytokines
    • TNF Superfamily
    • Signal Transduction
    • Growth Factors/Hormones
    • TNF
    • Stem Cells
    • Signaling Pathways
    • TGF beta
    • TNF alpha
    • Microbiology
    • Organism
    • Virus
    • RNA Virus
    • ssRNA positive strand virus
    • SARS Coronavirus
    • Cancer
    • Growth factors
    • TNF
    • Cancer
    • Tumor immunology
    • Cytokines
    • TNF
    • Cardiovascular
    • Atherosclerosis
    • Vascular Inflammation
    • Inflammatory mediators
    • Kits/ Lysates/ Other
    • Kits
    • ELISA Kits
    • ELISA Kits
    • TNF ELISA kits
    • Metabolism
    • Types of disease
    • Metabolic disorders
    • Neuroscience
    • Development
    • Neuroscience
    • Diseases
    • Neuroscience
    • Processes
  • 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
    modifications

    The 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.
  • Target information above from: UniProt accession P01375 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Alternative names

    • APC1
    • APC1 protein
    • Cachectin
    • DIF
    • Differentiation inducing factor
    • Macrophage cytotoxic factor
    • Tnf
    • TNF superfamily member 2
    • TNF superfamily, member 2
    • TNF, macrophage derived
    • TNF, monocyte derived
    • TNF-a
    • TNF-alpha
    • TNFA
    • TNFA_HUMAN
    • TNFSF2
    • Tumor necrosis factor
    • Tumor necrosis factor (TNF superfamily member 2)
    • Tumor necrosis factor alpha
    • Tumor necrosis factor ligand superfamily member 2
    • Tumor Necrosis Factor, Membrane Form
    • Tumor necrosis factor, soluble form
    see all
  • Database links

    • Entrez Gene: 7124 Human
    • Omim: 191160 Human
    • SwissProt: P01375 Human
    • Unigene: 241570 Human

    Associated products

    • Alternative Versions

      • Human TNF alpha Antibody Pair - BSA and Azide free (ab241791)
    • ELISA kits

      • Human TNF alpha ELISA Kit (ab100654)
      • Human TNF alpha ELISA Kit (ab108908)
      • Rat TNF alpha ELISA Kit (ab46070)
      • Human TNF alpha ELISA Kit (ab46087)
    • KO cell lines

      • Human TNF knockout THP-1 cell line (ab273761)
    • Related Products

      • (R,S)-Lenalidomide, Thalidomide analog, TNF alpha inhibitor (ab142129)

    Images

    • SimpleStep ELISA Protocol Diagram
      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.

       

    • Human TNF alpha standard curve comparison
      Human TNF alpha standard curve comparison

      Standard Curve comparison between human TNF alpha SimpleStep ELISA kit and traditional ELISA kit from leading competitor. SimpleStep ELISA kit shows increased sensitivity.

    • Example of human TNF alpha standard curve in Sample Diluent NS.
      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.

    • Raw data value for example of TNF standard curve in Sample Diluent NS
      Raw data value for example of TNF standard curve in Sample Diluent NS

      Raw data values are shown in the table

    • Interpolated concentrations of native TNF alpha in human PBMC cell culture supernatant samples.
      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.

    • Interpolated concentrations of native TNF alpha PBMC cell culture supernatant samples.
      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.

    • Linearity of dilution - native TNF alpha
      Linearity of dilution - native TNF alpha

      Native TNF alpha was measured in biological samples in a 2-fold dilution series. Sample dilutions are made in Sample Diluent NS.

    • Linearity of dilution - recombinant TNF alpha
      Linearity of dilution - recombinant TNF alpha

      Recombinant 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)
      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.”

    • Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421)
      Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421)
      To learn more about the advantages of recombinant antibodies see here.

    Protocols

    • Protocol Booklet

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (106)

    Publishing research using ab181421? Please let us know so that we can cite the reference in this datasheet.

    ab181421 has been referenced in 106 publications.

    • Lei Z  et al. Long non-coding RNA maternally expressed gene regulates cigarette smoke extract induced lung inflammation and human bronchial epithelial apoptosis via miR-149-3p. Exp Ther Med 21:60 (2021). PubMed: 33365060
    • Li N  et al. Astragaloside IV alleviates silica-induced pulmonary fibrosis via inactivation of the TGF-ß1/Smad2/3 signaling pathway. Int J Mol Med 47:N/A (2021). PubMed: 33448318
    • Zhang J  et al. IL-32 exacerbates adenoid hypertrophy via activating NLRP3-mediated cell pyroptosis, which promotes inflammation. Mol Med Rep 23:N/A (2021). PubMed: 33495843
    • Shi C  et al. lncRNA SNHG14 Plays a Role in Sepsis-Induced Acute Kidney Injury by Regulating miR-93. Mediators Inflamm 2021:5318369 (2021). PubMed: 33505213
    • Wang H  et al. HDAC1-mediated deacetylation of HIF1a prevents atherosclerosis progression by promoting miR-224-3p-mediated inhibition of FOSL2. Mol Ther Nucleic Acids 23:577-591 (2021). PubMed: 33510945
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
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    1-3 of 3 Abreviews or Q&A

    Works Perfect

    Excellent Excellent 5/5 (Ease of Use)
    Abreviews
    Abreviews
    Works as expected.
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Jul 15 2021

    TNF - CACO2 cells

    Excellent Excellent 5/5 (Ease of Use)
    Abreviews
    Abreviews
    abreview image
    We used this kit to detect TNF-a in Caco2 cell lines. The assay is easy to use and the protocol provided is detailed and well explicated. As you can see from the graph we got a nice standard curve and result (low error bars).
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    PROF. Michele D'angelo

    Verified customer

    Submitted Jan 15 2021

    efficient and accurate

    Excellent Excellent 5/5 (Ease of Use)
    Abreviews
    Abreviews
    I used this kit to detect TNF-a in stimulated human CD14+ monocytes. The assay was extremely efficient. There is no overnight coating step, and detection/capture antibodies are added as a cocktail at the same time as the samples. The assay was quite accurate, detecting even minimal amounts of TNF-a. I would use this assay again for its efficiency, requiring very little time at the bench.
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Jun 01 2016

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com

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