Human TNF alpha ELISA

RayBio® Human TNF alpha ELISA Kit for cell culture supernatants, plasma, serum, and lysate samples.

Lead time: Typically ships within 1-2 business days. No Friday shipments.

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Assay Format

Detection Range:
30 pg/ml - 6000 pg/ml
Recommended Dilution (serum & plasma):
2 fold
30 pg/ml

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This ELISA kit shows no cross-reactivity with any of the cytokines tested: Human ANG, CD23, Eotaxin, GCSF, GM-CSF, GRO-alpha, GRO-beta, GRO-gamma, I-309, IFN-gamma, IL-1 alpha, IL-1 beta, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-12 (p70), IL-15, IL-16, IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MCSF, MIG, MIP-1 alpha, MIP-1 beta, NAP-2, PDGF, PF-4
Number of Targets Detected:
Species Detected:
  • Human
Compatible Sample Types:
  • Cell Culture Supernatants
  • Cell Lysates
  • Plasma
  • Serum
  • Tissue Lysates
Design Principle:
  • Sandwich-based
Method of Detection:
  • Quantitative
Solid Support:
96-well Microplate

Product Specifications

1, 2, or 5 x 96-Well Strip Microplate Kit

Protein Information

Accession Number:
  • P01375
Gene ID:
  • 7124
Gene Symbols:
  • TNF
  • TNFA
  • TNFSF2
Protein Name & Synonyms:
Tumor necrosis factor (Cachectin) (TNF-alpha) (Tumor necrosis factor ligand superfamily member 2) (TNF-a) [Cleaved into: Tumor necrosis factor, membrane form (N-terminal fragment) (NTF) Intracellular domain 1 (ICD1) Intracellular domain 2 (ICD2) C-domain 1 C-domain 2 Tumor necrosis factor, soluble form]
Target Species:
  • Human

Target Background

Tumor necrosis factor alpha (TNF alpha), also known as lymphotoxin and cachectin, is a pleiotropic cytokine. TNF alpha is a smaller cytokine with a molecular weight of 26 kDa (transmembrane) and 17 kDa (soluble) that binds to the same receptors producing a vast array of effects similar to those of TNF-beta. Both TNF-alpha and TNF-beta share 30% amino acid homology and have similar biological activities. TNF-alpha is produced primarily by monocytes, neutrophils and macrophages. It is found in synovial cells and macrophages in the tissues. TNF-alpha and TNF-beta can modulate many immune and inflammatory functions, while having the ability to inhibit tumor growth. Target tumor cells must express TNF receptors 1 (55 kDa) and 2 (75 kDa) to be killed, with the p55 receptor mediating the cytotoxic response.

Product Features

Additional Notes
  • Strip plates and additional reagents allow for use in multiple experiments
  • Quantitative protein detection
  • Establishes normal range
  • The best products for confirmation of antibody array data

Standard Curves

Standard curve graphs showing the results from the human tnf alpha elisa kit

Dilution series diagram with 8 vials

Spiking & Recovery Results

Sample TypeAverage % RecoveryRange (%)
Cell culture media96.4384-104

Linearity Results

  SerumPlasmaCell Culture Media
1:2Average % of expected
Range (%)
1:4Average % of expected
Range (%)

Application Notes

Kit Components
  • Pre-Coated 96-well Strip Microplate
  • Wash Buffer
  • Stop Solution
  • Assay Diluent(s)
  • Lyophilized Standard
  • Biotinylated Detection Antibody
  • Streptavidin-Conjugated HRP
  • TMB One-Step Substrate
Other Materials Required
  • Distilled or deionized water
  • Precision pipettes to deliver 2 µl to 1 µl volumes
  • Adjustable 1-25 µl pipettes for reagent preparation
  • 100 µl and 1 liter graduated cylinders
  • Tubes to prepare standard and sample dilutions
  • Absorbent paper
  • Microplate reader capable of measuring absorbance at 450nm
  • Log-log graph paper or computer and software for ELISA data analysis
Protocol Outline
  1. Prepare all reagents, samples and standards as instructed in the manual.
  2. Add 100 µl of standard or sample to each well.
  3. Incubate 2.5 h at RT or O/N at 4°C.
  4. Add 100 µl of prepared biotin antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 µl of prepared Streptavidin solution to each well.
  7. Incubate 45 min at RT.
  8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 µl of Stop Solution to each well.
  11. Read at 450 nm immediately.

Need your results faster? Try RayBiotech's SpeedELISA platform for quantitative detection in just three hours.


The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.
  1. Genovese T, Impellizzeri D, D'Amico R, Fusco R, Peritore AF, Di Paola D, Interdonato L, Gugliandolo E, Crupi R, Di Paola R, Cuzzocrea S, Cordaro M, Siracusa R. Role of Bevacizumab on Vascular Endothelial Growth Factor in Apolipoprotein E Deficient Mice after Traumatic Brain Injury. Int J Mol Sci. 2022 Apr 9;23(8):4162. doi: 10.3390/ijms23084162. PMID: 35456980; PMCID: PMC9024601.
    Species: Mouse
    Sample type: Tissue Lysate
  2. Wang J, Han C, Lu Z, Ge P, Cui Y, Zhao D, Yang X, Wu B, Qiang L, Zhang Y, Chai Q, Lei Z, Li L, Hua Liu C, Zhang L. Simulated microgravity suppresses MAPK pathway-mediated innate immune response to bacterial infection and induces gut microbiota dysbiosis. FASEB J. 2020 Nov;34(11):14631-14644. doi: 10.1096/fj.202001428R. Epub 2020 Sep 12. PMID: 32918764.
    Species: Human
    Sample type: Conditioned media
  3. Sambataro M, Sambado L, Trevisiol E, Cacciatore M, Furlan A, Stefani PM, Seganfreddo E, Durante E, Conte S, Della Bella S, Paccagnella A, Dei Tos AP. Proinsulin-expressing dendritic cells in type 2 neuropathic diabetic patients with and without foot lesions. FASEB J. 2018 Jul;32(7):3742-3751. doi: 10.1096/fj.201701279RR. Epub 2018 Feb 12. PMID: 29436863.
    Species: Human
    Sample type: Plasma
  4. Yang, Y., Jiao, X., Li, L. et al. Increased Circulating Angiopoietin-Like Protein 8 Levels Are Associated with Thoracic Aortic Dissection and Higher Inflammatory Conditions. Cardiovasc Drugs Ther 34, 65–77 (2020).
    Species: Human
    Sample type: Conditioned media
  5. Joshi, I., Nazeer, R.A. Anti-inflammatory potential of novel hexapeptide derived from Meretrix meretrix foot and its functional properties. Amino Acids 52, 1391–1401 (2020).
    Species: Human
    Sample type: Conditioned media
  6. Dai, Y., Yan, T. & Gao, Y. Silence of miR-32-5p promotes endothelial cell viability by targeting KLF2 and serves as a diagnostic biomarker of acute myocardial infarction. Diagn Pathol 15, 19 (2020).
    Species: Human
    Sample type: Serum
  7. Chai, Q., Lu, Z., Liu, Z. et al. Lung gene expression signatures suggest pathogenic links and molecular markers for pulmonary tuberculosis, adenocarcinoma and sarcoidosis. Commun Biol 3, 604 (2020).
    Species: Human
    Sample type: Conditioned media
  8. Piano S, Favaretto E, Tonon M, Antonelli G, Brocca A, Sticca A, Mareso S, Gringeri E, Scaroni C, Plebani M, Russo FP, Burra P, Cillo U, Angeli P. Including Relative Adrenal Insufficiency in Definition and Classification of Acute-on-Chronic Liver Failure. Clin Gastroenterol Hepatol. 2020 May;18(5):1188-1196.e3. doi: 10.1016/j.cgh.2019.09.035. Epub 2019 Oct 4. PMID: 31589973.
    Species: Human
    Sample type: Plasma, Saliva
  9. Guerrero CLH, Yamashita Y, Miyara M, Imaizumi N, Kato M, Sakihama S, Hayashi M, Miyagi T, Karimata K, Uchihara J, Ohshiro K, Todoroki J, Nakachi S, Morishima S, Karube K, Tanaka Y, Masuzaki H, Fukushima T. Proteomic profiling of HTLV-1 carriers and ATL patients reveals sTNFR2 as a novel diagnostic biomarker for acute ATL. Blood Adv. 2020 Mar 24;4(6):1062-1071. doi: 10.1182/bloodadvances.2019001429. Erratum in: Blood Adv. 2020 Apr 28;4(8):1647. PMID: 32196559; PMCID: PMC7094015.
    Species: Human
    Sample type: Plasma
  10. Chatterjee S, Bhattcharjee D, Misra S, Saha A, Bhattacharyya NP, Ghosh A. Increase in MEG3, MALAT1, NEAT1 significantly predicts the clinical parameters in patients with rheumatoid arthritis. Per Med. 2020 Nov;17(6):445-457. doi: 10.2217/pme-2020-0009. Epub 2020 Oct 7. PMID: 33026292.
    Species: Human
    Sample type: Serum
Excellent performance for the assessment of THP-1 polarization towards pro-inflammatory macrophages.

University of Puerto Rico at Maya
Excellent product, I used it for a THP inhibitory assay with great accuracy and reproducibility.

University of Puerto Rico
Straightforward protocol!

Great standard curve! I used on supernatants and no needed to dilute my samples (PASMCs medium).
Highly recommended

This ELISA kit was used for quantification of TNF-alpha in the supernatants of SEB-stimulated peripheral blood mononuclear cells in combination with conventional chemotherapeutics. Overall, the assay worked well, and the output was clear.

Worked great with THP-1 cells stimulated with LPS. Sensitive and easy to use.

Duke University
Great kit. Easy to follow protocol. Getting great results.

Florida A&M University
It's so easy to use. I'm happy with it and recommend it to everyone.

Columbia University
Simple protocol and reproducible results. Would recommend.

University of Houston
I used it for human corneal epithelial cell culture supernatant under induced conditions. It is very sensitive and easy to use.

I also want to comment on the fast delivery of the product.

University of Illinois at Chicago
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This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.

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