TNF ELISA Kit (Human) (OKBB00261)

Catalog No: OKBB00261

Size:96 Tests

Human TNF ELISA Kit standard curve

Product Info

• Predicted Species Reactivity: Human
• Application: ELISA
• ELISA Kit Detection Method: Colorimetric, OD450 nm
• ELISA Kit Duration: ~ 3 Hours
• ELISA Kit Principle: Aviva's human TNF alpha ELISA Kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. A monoclonal antibody from mouse specific for TNF alpha has been precoated onto 96-well plates. Standards (E.coli,V77-L233) and test samples are added to the wells, a biotinylated detection polyclonal antibody from goat specific for TNF alpha is added subsequently and then followed by washing with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex was added and unbound conjugates were washed away with PBS or TBS buffer. HRP substrate TMB was used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The absorbance of yellow is proportional to the human TNF alpha amount of sample captured in plate.
• ELISA Kit Range: 15.6 pg/ml - 1,000 pg/ml
• ELISA Kit Reproducibility:

  	Intra-Assay Precision			Inter-Assay Precision
  Sample	1	2	3	1	2	3
  Mean (pg/mL)	93	327	608	102	319	613
  St. Dev.	5.1	15.4	31	7.65	15.3	35
  %CV	5.48	4.70	5.09	7.5	4.79	5.70

• ELISA Kit Component:
  • Lyophilized recombinant human TNFalpha standard: 10 ng/tube x 2
  • Anti-human TNFalpha Antibody Well Plate: 96 Wells
  • Sample diluent buffer: 30 mL
  • Biotinylated anti-human TNF antibody: 130 uL, dilution 1:100
  • Antibody diluent buffer: 12 mL
  • Avidin-Biotin-Peroxidase Complex (ABC): 130 uL, dilution 1:100
  • ABC diluent buffer: 12 mL
  • TMB color developing agent: 10 mL
  • TMB stop solution: 10 mL
• Additional Information: Range: 15.6pg/ml-1000pg/ml (cell culture supernates),7.8pg/ml-500pg/ml (serum, plasma)
  Principle: Aviva’s human TNFα ELISA Kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. A monoclonal antibody from mouse specific for TNFα has been precoated onto 96-well plates. Standards (E.coli,V77-L233) and test samples are added to the wells, a biotinylated detection polyclonal antibody from goat specific for TNFα is added subsequently and then followed by washing with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex was added and unbound conjugates were washed away with PBS or TBS buffer. HRP substrate TMB was used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to thehuman TNFα amount of sample captured in plate.
  Notes: 1. To inspect the validity of experiment operation and the appropriateness of sample dilution proportion, pilot
  experiment using standards and a small number of samples is recommended.
  2. The TMB Color Developing agent is colorless and transparent before using, contact us freely if it is not the case.
  3. Before using the Kit, spin tubes and bring down all components to the bottom of tubes.
  4. Duplicate well assay is recommended for both standard and sample testing.
  5. Don’t let 96-well plate dry, for dry plate will inactivate active components on plate.
  6. Don’t reuse tips and tubes to avoid cross contamination.
  7. To avoid to use the reagents from different batches together.
  8. In order to avoid marginal effect of plate incubation due to temperature difference ( reaction may be stronger in the
  marginal wells), it is suggested that the diluted ABC and TMB solution will be pre-warmed in 37C for 30 min before
  using.
  Background: Tumor necrosis factor-alpha (TNF-alpha, or TNF) is secreted by macrophages in response to inflammation, infection and cancer.1 Human Tumor Necrosis Factor (TNF) and Lymphotoxin (TNF-beta) are cytotoxic proteins which have similar biological activities and share 30% amino acid homology.2 TNF-alpha is produced by monocytes, which can stimulate endothelial cells to produce the multilineage growth factor granulocyte-macrophage colony-stimulating factor and extend the role of this immunoregulatory protein to the regulation of hematopoiesis in vitro.3 TNF is a soluble protein that causes damage to tumor cells but has no effect on normal cells. Human TNF has been purified to apparent homogeneity as a 17.3-kilodalton protein from HL-60 leukemia cells and has showed cytotoxic and cytostatic activities against various human tumor cell lines. The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids. The mature protein begins at residue 77, leaving a long leader sequence of 76 amino acids.4 TNF-alpha has been mapped to human chromosome 6.2
• Reconstitution and Storage: Store at 4C for 6 months, at -20C for 12 months. Avoid multiple freeze-thaw cycles (Shipped with wet ice.)
• Sample Type: Cell culture supernates, serum, and plasma (heparin, EDTA, citrate)
• Sensitivity: <1 pg/ml
• Reference: 1. Brenner, D. A.; O'Hara, M.; Angel, P.; Chojkier, M.; Karin, M. Prolonged activation of JUN and collagenase genes by tumour necrosis factor-alpha. Nature 337: 661-663, 1989.
  2. Nedwin, G. E.; Naylor, S. L.; Sakaguchi, A. Y.; Smith, D.; Jarrett-Nedwin, J.; Pennica, D.; Goeddel, D. V.; Gray, P. W. Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization. Nucleic Acids Res. 13: 6361-6373, 1985.
  3. Broudy, V. C.; Kaushansky, K.; Segal, G. M.; Harlan, J. M.; Adamson, J. W. Tumor necrosis factor type alpha stimulates human endothelial cells to produce granulocyte/macrophage colony-stimulating factor. Proc. Nat. Acad. Sci. 83: 7467-7471, 1986.
  4. Wang, A. M.; Creasey, A. A.; Ladner, M. B.; Lin, L. S.; Strickler, J.; Van Arsdell, J. N.; Yamamoto, R.; Mark, D. F. Molecular cloning of the complementary DNA for human tumor necrosis factor. Science 228: 149-154, 1985.
• Specificity: Natural and recombinant human TNF alpha
• Immunogen: Expression system for standard: E.coli; Immunogen sequence: V77-L233
• Description: For quantitative detection of human TNF alpha in cell culture supernatants, serum and plasma(heparin, EDTA, citrate).

Target Info

• Gene Symbol: TNF
• Gene Full Name: tumor necrosis factor
• Alias Symbols: APC1 protein, Cachectin, DIF, soluble form, Tnf, TNF a, TNF alpha, TNF macrophage derived, TNF monocyte derived, TNF superfamily member 2, TNF-a, TNF-alpha, TNFA_HUMAN, TNFSF2, Tumor necrosis factor, Tumor necrosis factor alpha, Tumor necrosis factor ligand superfamily member 2
• NCBI Gene Id: 7124
• Protein Name: Tumor necrosis factor
• Description of Target: Tumor necrosis factor-alpha (TNF-alpha, or TNF) is secreted by macrophages in response to inflammation, infection and cancer.1 Human Tumor Necrosis Factor (TNF) and Lymphotoxin (TNF-beta) are cytotoxic proteins which have similar biological activities and share 30% amino acid homology.2 TNF-alpha is produced by monocytes, which can stimulate endothelial cells to produce the multilineage growth factor granulocyte-macrophage colony-stimulating factor and extend the role of this immunoregulatory protein to the regulation of hematopoiesis in vitro.3 TNF is a soluble protein that causes damage to tumor cells but has no effect on normal cells. Human TNF has been purified to apparent homogeneity as a 17.3-kilodalton protein from HL-60 leukemia cells and has showed cytotoxic and cytostatic activities against various human tumor cell lines. The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids. The mature protein begins at residue 77, leaving a long leader sequence of 76 amino acids.4 TNF-alpha has been mapped to human chromosome 6.
• Uniprot ID: P01375
• Protein Accession #: NP_000585.2
• Nucleotide Accession #: NM_000594.3