Human TNF-alpha ELISA - Quantikine QuicKit Best Seller
R&D Systems, part of Bio-Techne | Catalog # QK210
Key Product Details
Assay Length
80 minutes
Sample Type & Volume Required Per Well
Cell Culture Supernates (50 µL), Serum (50 µL), EDTA Plasma (50 µL), Heparin Plasma (50 µL)
Sensitivity
7.02 pg/mL
Assay Range
31.3-2000 pg/mL (Cell Culture Supernates, Serum, EDTA Plasma, Heparin Plasma)
Product Summary for Human TNF-alpha Quantikine QuicKit ELISA
The Quantikine® QuicKit™ Human TNF-alpha Immunoassay is a one step, 80 minute solid phase ELISA designed to measure human TNF-alpha in cell culture supernates, serum, and plasma. It contains E.
coli-expressed recombinant human TNF-alpha and antibodies raised against the recombinant protein. Results obtained for naturally occurring human TNF-alpha showed linear curves that were parallel to the standard curves obtained using the recombinant QuicKit™ standards. These results indicate that this kit can be used to determine relative mass values for natural human TNF-alpha.
Product Specifications
Measurement
Quantitative ELISA
Detection Method
Colorimetric - 450nm (TMB)
Conjugate
HRP
Reactivity
Human
Specificity
Natural and recombinant human TNF-alpha.
Cross-reactivity
< 0.5% cross-reactivity observed with available related molecules. < 50% cross-species reactivity observed with species tested.
Interference
No significant interference observed with available related molecules.
Sample Values
Serum/Plasma - Ten serum and plasma samples from apparently healthy volunteers
were evaluated for the presence of human TNF-alpha in this assay. All samples measured less than
the lowest standard, 31.3 pg/mL. No medical histories were available for the donors used in
this study.
Cell Culture Supernates - Human peripheral blood mononuclear cells (PBMCs)
(1 x 106
cells/mL) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum,
2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. Cells were
unstimulated or stimulated with 10 μg/mL of PHA for 5 days. Aliquots of the culture supernates
were removed, assayed for levels of human TNF-alpha, and measured 80.9 pg/mL and 9317 pg/mL,
respectively.
Human monocyte-derived macrophages (MDM) were obtained from PBMCs and attached
monocytes were cultured in StemXVivo Serum-free Dendritic Cell Base Media (R&D Systems,
Catalog#, CCM003) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin sulfate,
and 100 ng/mL of recombinant human M-CSF for 5 days. Cells were unstimulated or stimulated
with 1 μg/mL LPS and 40 ng/mL of recombinant human IFN-gamma for 24 hours. Aliquots of the cell
culture supernates were removed, assayed for levels of human TNF-alpha, and measured
40.5 pg/mL and 6482 pg/mL, respectively.
Precision
Intra-Assay Precision (Precision within an assay) Two samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays) Two samples of known concentration were tested in ten separate assays to assess inter-assay precision. Assays were performed by at least three technicians.
Cell Culture Supernates, EDTA Plasma, Heparin Plasma, Serum
| Intra-Assay Precision | Inter-Assay Precision | |||
|---|---|---|---|---|
| Sample | 1 | 2 | 1 | 2 |
| n | 20 | 20 | 10 | 10 |
| Mean (pg/mL) | 200 | 1177 | 214 | 1243 |
| Standard Deviation | 3.64 | 28.5 | 13.9 | 67.8 |
| CV% | 1.8 | 2.4 | 6.5 | 5.5 |
Recovery for Human TNF-alpha Quantikine QuicKit ELISA
The recovery of human TNF-alpha spiked to three different levels in samples throughout the range of the assay in various matrices was evaluated.
| Sample Type | Average % Recovery | Range % |
|---|---|---|
| Cell Culture Media (n=4) | 120 | 108-126 |
| EDTA Plasma (n=2) | 95 | 87-106 |
| Heparin Plasma (n=2) | 89 | 81-103 |
| Serum (n=2) | 86 | 83-88 |
Linearity
To assess linearity of the assay, samples containing and/or spiked with high concentrations of human TNF-alpha in various matrices were diluted with the calibrator diluent to produce samples with values within the dynamic range of the assay.
Scientific Data Images for Human TNF-alpha Quantikine QuicKit ELISA
Human TNF- alpha Standard Curve
Human TNF- alpha Standard Curve
Human TNF-alpha ELISA Standard Curve
Preparation and Storage
Shipping
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Background: TNF-alpha
Human TNF-alpha is synthesized as a 26 kDa type II transmembrane protein that consists of
a 35 amino acid (aa) cytoplasmic domain, a 21 aa transmembrane segment, and a 177 aa
extracellular domain (ECD) (12, 13). Within the ECD, human TNF-alpha shares 97% aa sequence
identity with rhesus monkey, and 71%-92% aa identity with bovine, canine, cotton rat, equine,
feline, mouse, porcine, and rat TNF-alpha. It is produced by a wide variety of immune, epithelial,
endothelial, and tumor cells. TNF-alpha is assembled intracellularly to form a noncovalently linked
homotrimer which is expressed on the cell surface (14). Cell surface TNF-alpha can both induce the
lysis of tumor cells and virus infected cells, and generate its own downstream cell signaling
following ligation by soluble TNF RI (15, 16). Shedding of membrane bound TNF-alpha by TACE/
ADAM17 releases the bioactive cytokine, a 55 kDa soluble trimer of the TNF-alpha extracellular
domain (17-19).
TNF-alpha binds the ubiquitous 55-60 kDa TNF RI (20, 21) and the hematopoietic cell-restricted
78-80 kDa TNF RII (22, 23), both of which are also expressed as homotrimers (1, 24). Both
type I and type II receptors bind TNF-alpha with comparable affinity and can promote NF kappaB
activation (25-28). Only TNF RI, however, contains a cytoplasmic death domain which triggers
the activation of apoptosis (3, 29). Soluble forms of both types of receptors are released into
human serum and urine and can neutralize the biological activity of TNF-alpha (30-32).
Long Name
Tumor Necrosis Factor alpha
Alternate Names
Cachetin, DIF, TNF, TNF-A, TNFA, TNFalpha, TNFG1F, TNFSF1A, TNFSF2
Entrez Gene IDs
Gene Symbol
TNF
Additional TNF-alpha Products
Product Documents for Human TNF-alpha Quantikine QuicKit ELISA
Product Specific Notices for Human TNF-alpha Quantikine QuicKit ELISA
For research use only
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