| Test principle: |
This assay employs a two-site sandwich ELISA to quantitate TNF-α in samples. An antibody specific for TNF-α has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any TNF-α present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for TNF-α is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of TNF-α bound in the initial step. The color development is stopped and the intensity of the color is measured.
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| Product Overview: |
TNFa is synthesized as a 26 kDa, type II transmembrane protein that is 233 amino acids in length. It contains a 30 amino acid (aa) cytoplasmic domain, a 26 aa transmembrane segment, and a 177 aa extracellular region. TNFa is assembled intracellularly to form a transmembrane, non-covalently-linked homotrimeric protein. The 157 aa residue soluble form of TNFa (sTNF-αis released from the C-terminus of the transmembrane protein through the activity of TNFa-converting enzyme (TACE), a membrane bound disintegrin metalloproteinase. Rat cells known to express TNF-αinclude B cells, colonic columnar epithelial cells, NK and CD3 CD56 hepatic natural T cells, macrophages, monocytes and monocyte-derived dendritic cells, CD4 and CD8 T cells, mast cells, neutrophils, keratinocytes, plasma cells, and adipocytes.
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| Components: |
Reagents |
Quantity |
Reagents |
Quantity |
Assay plate (96 Wells) |
1 |
Instruction manual |
1 |
Standard (lyophilized) |
2 |
Sample Diluent |
1 x 20 mL |
Biotin-Conjugate (concentrate 100 x) |
1 x 120 μL |
Biotin-Conjugate Diluent |
1 x 20 mL |
Streptavidin-HRP (concentrate 100 x) |
1 x 120 μL |
Streptavidin-HRP Diluent |
1 x 20 mL |
Wash Buffer (concentrate 25 x) |
1 x 20 mL |
Substrate Solution |
1 x 10 mL |
Stop Solution |
1 x 10 mL |
Adhesive Films |
4 |
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| Specificity: |
This assay has high sensitivity and excellent specificity for detection of Rabbit TNF-α. No significant cross-reactivity or interference between Rabbit TNF-α and analogues was observed.
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| Recovery: |
Matrices listed below were spiked with certain level of recombinant Rabbit TNF-α and the recovery rates were calculated by comparing the measured value to the expected amount of Rabbit TNF-α in samples.
Sample Type |
Number |
Recovery range (%) |
Average(%) |
Serum |
10 |
90-101 |
96 |
EDTA plasma |
10 |
89-97 |
93 |
Heparin plasma |
10 |
91-99 |
95 |
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| Precision: |
Intra-assay Precision (Precision within an assay)
Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-assay Precision (Precision between assays)
Three samples of known concentration were tested in forty separate assays to assess inter-assay precision.
CV (%) = SD/meanX100
Intra-Assay: CV<8%
Inter-Assay: CV<12%
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| Linearity: |
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rabbit TNF-α and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample Type |
1：2 |
1：4 |
1：8 |
1：16 |
Serum |
78-89% |
81-99% |
92-103% |
95-105% |
EDTA plasma |
91-101% |
90-98% |
93-101% |
91-98% |
Heparin plasma |
92-103% |
93-102% |
92-99% |
91-101% |
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| Stability: |
The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.
The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).
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| Sample collection and storage: |
Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles.
Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles.
Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
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| Kits storage instructions: |
Store at 2-8°C. Please refer to Instruction Manual.
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Rabbit Tumor necrosis factor α (TNF-α) ELISA Kit
