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The Human TNF alpha ELISA kit is a single-wash 90-min SimpleStep ELISA® used to quantify Human TNF alpha with a sensitivity of 4.32 pg/ml. The assay uses a simple Mix-Wash-Read protocol with just one incubation and wash step.Easy-to-use, rapid, sensitive human TNF alpha sandwich ELISA kit:- Colorimetric assay - 450nm readout; works on any plate reader- Different formats for different needs: 10x96 plates for bulk orders and 384-well for higher throughput- Broad sample compatibility – works with serum, plasma, cell culture supernatants, and other biological fluids


Images

Sandwich ELISA - Human TNF alpha ELISA Kit (AB181421), expandable thumbnail
  • Sandwich ELISA - Human TNF alpha ELISA Kit (AB181421), expandable thumbnail
  • Sandwich ELISA - Human TNF alpha ELISA Kit (AB181421), expandable thumbnail
  • Sandwich ELISA - Human TNF alpha ELISA Kit (AB181421), expandable thumbnail
  • Sandwich ELISA - Human TNF alpha ELISA Kit (AB181421), expandable thumbnail

Publications

Key facts

Detection method

Colorimetric

Sample types

Cerebral Spinal Fluid, Heparin Plasma, Citrate plasma, Cell culture supernatant, Serum, EDTA Plasma

Assay type

Sandwich (quantitative)

Reactive species

Human

Range

15.63 - 1000 pg/mL

Assay time

1h 30m

Sensitivity

= 4.32 pg/mL

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Reactivity data

Application

sELISA

Reactivity

Reacts

Dilution info
-
Notes

-

Associated Products

Select an associated product type

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Target data

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. Impairs regulatory T-cells (Treg) function in individuals with rheumatoid arthritis via FOXP3 dephosphorylation. Up-regulates the expression of protein phosphatase 1 (PP1), which dephosphorylates the key 'Ser-418' residue of FOXP3, thereby inactivating FOXP3 and rendering Treg cells functionally defective (PubMed:23396208). Key mediator of cell death in the anticancer action of BCG-stimulated neutrophils in combination with DIABLO/SMAC mimetic in the RT4v6 bladder cancer cell line (PubMed:16829952, PubMed:22517918, PubMed:23396208). Induces insulin resistance in adipocytes via inhibition of insulin-induced IRS1 tyrosine phosphorylation and insulin-induced glucose uptake. Induces GKAP42 protein degradation in adipocytes which is partially responsible for TNF-induced insulin resistance (By similarity). Plays a role in angiogenesis by inducing VEGF production synergistically with IL1B and IL6 (PubMed:12794819). Promotes osteoclastogenesis and therefore mediates bone resorption (By similarity).The TNF intracellular domain (ICD) form induces IL12 production in dendritic cells.

Alternative names

What's included?

1 x 384 Tests
Components
10X Wash Buffer PT (ab206977)
1 x 20 mL
384 well CaptSure™ microplates
1 x 1 Unit
Antibody Diluent 4BR
1 x 6 mL
Human TNFa Capture Antibody
1 x 600 µL
Human TNFa Detector Antibody
1 x 600 µL
Human TNFa Lyophilized Recombinant Protein
2 x 1 Vial
Plate Seals
1 x 1 Unit
Sample Diluent NS (ab193972)
2 x 50 mL
Stop Solution
2 x 12 mL
TMB Development Solution
2 x 12 mL

Recommended products

The Human TNF alpha ELISA kit is a single-wash 90-min SimpleStep ELISA® used to quantify Human TNF alpha with a sensitivity of 4.32 pg/ml. The assay uses a simple Mix-Wash-Read protocol with just one incubation and wash step.Easy-to-use, rapid, sensitive human TNF alpha sandwich ELISA kit:- Colorimetric assay - 450nm readout; works on any plate reader- Different formats for different needs: 10x96 plates for bulk orders and 384-well for higher throughput- Broad sample compatibility – works with serum, plasma, cell culture supernatants, and other biological fluids

Key facts

Detection method

Colorimetric

Sample types

Cerebral Spinal Fluid, Heparin Plasma, Citrate plasma, Cell culture supernatant, Serum, EDTA Plasma

Assay type

Sandwich (quantitative)

Reactive species

Human

Range

15.63 - 1000 pg/mL

Assay time

1h 30m

Assay Platform

Microplate

Sensitivity

= 4.32 pg/mL

Precision

Intra assay

Sample

PBMC media

n
8
mean
-
SD
-
C.V.

2.5

Inter assay

Sample

PBMC media

n
3
mean
-
SD
-
C.V.

3.1

Recovery

Sample specific recovery

Sample type

Cerebral Spinal Fluid

Average %

= 83

Range

80 - 88 %

Sample type

Cell culture supernatant

Average %

= 110

Range

105 - 113 %

Sample type

Serum

Average %

= 88

Range

83 - 93 %

Sample type

EDTA Plasma

Average %

= 100

Range

95 - 104 %

Sample type

Heparin Plasma

Average %

= 98

Range

89 - 110 %

Sample type

Citrate plasma

Average %

= 91

Range

86 - 97 %

Storage

Shipped at conditions

Blue Ice

Appropriate short-term storage conditions

+4°C

Appropriate long-term storage conditions

+4°C

Storage information

+4°C

Notes

Human TNF alpha ELISA Kit ab181421 is a rapid single-wash 90-min ELISA kit to measure Human TNF alpha in cerebral spinal fluid (CSF), cell culture supernatant, serum and plasma. This SimpleStep ELISA® sensitivity is 4.32 pg/mL.

How the assay works

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.

Assay protocol summary

Human TNF alpha ELISA Kit ab181421 protocol summary:

1. Mix : add samples / standards to the wells together with the capture and detector antibody cocktail. Incubate 1 hour at room temperature
2. Wash
3. Add TMB development solution - incubate for 10 min
4. Add Stop solution
5. Read the results on a plate reader at 450 nm

Alternative assay kits

Need to design your own immunoassay? We offer the antibody pair used in this kit in a BSA and Azide-free format, ready for conjugation:

- Human/Monkey TNF alpha Antibody Pair - BSA and Azide free Human/Monkey TNF alpha Antibody Pair - BSA and Azide free ab241791,
- Anti-TNF alpha antibody [EPR18565-29] - BSA and Azide free (Capture) Anti-TNF alpha antibody [EPR18565-29] - BSA and Azide free (Capture) ab242476,
- Anti-TNF alpha antibody [EPR18565-33] - BSA and Azide free (Detector) Anti-TNF alpha antibody [EPR18565-33] - BSA and Azide free (Detector) ab242978

Getting the best performance

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).

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.

Save your precious samples

Save your precious samples: our 384-well Human TNF alpha ELISA Kit only requires a maxmum of 12.5µL

How other researchers are using this kit

Human TNF alpha ELISA Kit ab181421 has been used to assess inflammation in different physio-pathological contexts such as obesity (1), coronary artery disease (2), and rheumatoid arthritis (3).

References: 1-Shaohong Yu et al. 2023, PMID: 38156278, 2-Yongshun Wang et al. 2023, PMID: 38054817, 3-Yaju Tan et al. 2024, PMID: 37997635

Related and recommended products

Human TNF alpha ELISA kit ab181421 is commonly used to measure TNF alpha as a marker of inflammation.

Other ELISA kits to measure inflammatory markers include:

- Human IL-6 ELISA kit Human IL-6 ELISA Kit ab178013
- Human IL-8 ELISA kit Human IL-8 ELISA Kit ab214030
- Human IL-1 beta ELISA kit Human IL-1 beta ELISA Kit ab214025

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.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

Tumor necrosis factor alpha (TNF alpha) also known as TNF-a is a cytokine with a molecular weight of approximately 17 kDa. This protein is expressed mainly by macrophages though other cells like T-cells and natural killer cells also produce it. TNF alpha helps in regulating immune responses and inflammatory reactions. Its expression is important in triggering cell apoptosis and modulating immune cell activities. TNF alpha exists in membrane-bound and soluble forms both having biologic activity.

Biological function summary

TNF alpha acts as an important regulator of inflammatory and immune responses. It is not part of a complex but interacts with TNF receptors to activate various signaling cascades. TNF alpha promotes leukocyte recruitment to sites of infection or injury by enhancing adhesion molecule expression on endothelial cells. Additionally it induces the production of other pro-inflammatory cytokines like IL-6 and IL-1 amplifying the immune response. Researchers often measure TNF alpha levels in samples using methods like ELISA or enzyme-linked immunosorbent assay and develop specific antibodies like anti-TNF alpha for experimental and therapeutic purposes.

Pathways

TNF alpha participates in the NF-kB and MAPK pathways significant for mediating inflammatory and immune responses. In the NF-kB pathway TNF alpha activates transcription factors that control the expression of genes involved in inflammation and cell survival. TNF alpha also interacts with proteins such as the TNF receptor 1 (TNFR1) and receptor-associated factors (TRAFs) further relaying signals within these pathways. There is extensive research employing animal models using mouse TNF-alpha and anti-TNF reagents to dissect these pathways’ intricacies.

Associated diseases and disorders

TNF alpha substantially affects conditions like rheumatoid arthritis and Crohn's disease both characterized by chronic inflammation. Elevated levels of TNF alpha drive the inflammatory processes central to these diseases leading to tissue damage. Other proteins like IL-6 also play critical roles in these disorders and together with TNF alpha create a pro-inflammatory environment. Therapeutic agents such as anti-TNF treatments aim to mitigate these effects by specifically targeting TNF alpha activity.

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9 product images

  • Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421), expandable thumbnail

    Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421)

    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.

  • Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421), expandable thumbnail

    Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421)

    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.

  • Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421), expandable thumbnail

    Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421)

    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.

  • Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421), expandable thumbnail

    Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421)

    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.

  • Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421), expandable thumbnail

    Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421)

    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.

  • Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421), expandable thumbnail

    Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421)

    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), expandable thumbnail

    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 (Human TNF knockout THP-1 cell line 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), expandable thumbnail

    Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421)

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

    Raw data values are shown in the table

  • Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421), expandable thumbnail

    Sandwich ELISA - Human TNF alpha ELISA Kit (ab181421)

    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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For this product, it's our understanding that no specific protocols are required. You can:

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