Human TNF RI/TNFRSF1A Quantikine QuicKit ELISA

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QK225
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Human TNF RI ELISA Standard Curve
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Human TNF RI/TNFRSF1A Quantikine QuicKit ELISA Summary

Assay Length
80 minutes
Sample Type & Volume Required Per Well
Cell Culture Supernates (50 uL), Serum (13 uL), EDTA Plasma (13 uL), Heparin Plasma (13 uL), Urine (13 uL)
Sensitivity
1.69 pg/mL
Assay Range
15.6 - 1,000 pg/mL (Cell Culture Supernates, Serum, EDTA Plasma, Heparin Plasma, Urine)
Specificity
Natural and recombinant human TNF RI.
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.

Product Summary

The Quantikine® QuicKit™ Human TNF RI/TNFRSF1A Immunoassay is a one step, 80-minute solid phase ELISA designed to measure human TNF RI levels in cell culture supernates, serum, plasma, and urine. It contains E. coli-expressed recombinant human TNF RI and antibodies raised against the recombinant protein. Results obtained for natural human TNF RI 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 RI.

Precision

Intra-Assay Precision (Precision within an assay) <div>Two samples of known concentration were tested twenty times on one plate to assess&nbsp;intra-assay precision.</div>
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, Serum, EDTA Plasma, Heparin Plasma, Urine

Intra-Assay Precision Inter-Assay Precision
Sample 1 2 1 1
n 20 20 10 10
Mean 100 624 619 99.9
Standard Deviation 1.75 17 39.3 6.78
CV% 1.8 2.7 6.3 6.8

Recovery

The recovery of human TNF RI 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) 101 92-109
EDTA Plasma (n=2) 98 88-109
Heparin Plasma (n=2) 98 85-109
Serum (n=2) 99 91-113
Urine (n=2) 96 92-100

Linearity

To assess the linearity of the assay, samples containing and/or spiked with high concentrations of human TNF RI were diluted with calibrator diluent to produce samples with values within the dynamic range of the assay.
Human TNF RI ELISA Linearity

Data Examples

Human TNF RI ELISA Standard Curve

Human TNF RI QuicKit Spiked Recovery Competitor Comparison TNF RI is spiked at three known concentrations throughout the range of the assay and run to measure response of the spiked sample matrix. Serum recovery is 108% compared to 81% for the top competitor. Plasma recovery is 82% compared to 75% for the top competitor. Urine recovery is 92% compared to 83% for the top competitor. In spike and recovery experiments, natural samples are spiked with the recombinant target analyte of interest to identify interference caused by sample matrices.

Human TNF RI QuicKit Natural Linearity Competitor Comparison Samples containing TNF RI were diluted with appropriate Calibrator Diluent to produce samples with values within the dynamic range of the assay. The linearity is between 108%-117% compared to 113%-164% for the top competitor.

Product Datasheets

Preparation and Storage

Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: TNF RI/TNFRSF1A

Tumor necrosis factors (TNFs) are pleiotropic cytokines that are considered primary modifiers of the inflammatory and immune reactions of animals produced in response to injury or infection. Two forms of TNF, designated TNF-alpha (or cachectin) and TNF-beta (or lymphotoxin), have been described that share 30% sequence similarity and compete for binding to the same receptors. TNFs play a necessary and beneficial role as mediators of host resistance to infections and tumor formation. However, over-production or inappropriate expression of these factors can lead to a variety of pathological conditions, including wasting, systemic toxicity, and septic shock. For reviews of the literature relating to these factors, see references 1 and 2. 

The actions of TNFs are produced subsequent to binding of the factors to cell surface receptors. Two distinct TNF receptors have been identified and cloned. Virtually all cell types studied show the presence of one or both of these receptor types. One receptor type, termed TNF RII (Type A, Type a, 75 kDa or utr antigen), shows an apparent molecular weight of 75 kDa. The gene for this receptor encodes a presumptive transmembrane protein of 439 amino acid (aa) residues (3, 19). The other receptor type, termed TNF RI (Type B, Type b, 55 kDa or htr antigen), shows an apparent molecular weight of 55 kDa. The gene for this protein encodes a transmembrane protein of 426 aa residues (4, 5, 19). Both receptor types show high affinity binding of either TNF-alpha or TNF-beta. The two receptor types are immunologically distinct but their extracellular domains show similarities in the pattern of cysteine residue locations in four domains (3). The intracellular domains of the two receptor types are apparently unrelated, suggesting the possibility that the two receptor types employ different signal transduction pathways. 
Several groups have identified soluble TNF binding proteins in human serum and urine (6-8) that can neutralize the biological activities of TNF-alpha and TNF-beta. Two types have been identified and designated sTNF RI (or TNF BPI) and sTNF RII (or TNF BPII). These soluble forms have now been shown to represent truncated forms of the two types of TNF receptors discussed above. The soluble receptor forms apparently arise as a result of shedding of the extracellular domains of the receptors, and concentrations of about 1-2 ng/mL are found in the serum and urine of healthy subjects (9, 10). The levels of the soluble receptors vary from individual to individual but are stable over time for given individuals (9). 
Elevated levels of TNF receptors have been found in the amniotic fluid and urine of pregnant women (11), in serum or plasma in association with pathological conditions such as endotoxinemia (12, 13), meningiococcemia (14), and HIV infection (15), and in plasma and ascites of patients in association with infections and malignancies (16). The mechanisms involved in the induction of shedding of the TNF receptors are not well understood. There are reports of correlations between increased TNF levels and soluble receptor levels, suggesting generally that stimuli that cause TNF levels to rise also induce shedding of TNF receptors (12-14, 17). There is also evidence, however, that suggests the shedding of the two types of soluble receptors is independently regulated (13). 2 For research use only. Not for use in diagnostic procedures. 
The physiological role of the soluble TNF receptors is not known. It is known that both types of soluble receptors can bind to TNF in vitro and inhibit its biological activity by competing with cell surface receptors for TNF binding. Consequently it has been suggested that shedding of soluble receptors in response to TNF release could serve as a mechanism for binding and inhibiting the TNF not immediately bound to surface receptors, thus protecting other cells from the effects of TNF and localizing the inflammatory response (12, 17). It is also possible that shedding of receptors represents a mechanism for desensitizing the cells that shed the receptors from the effects of TNF (17). On the other hand, it has been reported that at low concentrations of TNF, binding to soluble receptors can stabilize TNF and augment some of its activities (18). Thus it is possible that under some conditions the pool of TNF bound to soluble receptors could represent a reservoir for the stabilization and controlled release of TNF.

Long Name:
Tumor Necrosis Factor Receptor I
Entrez Gene IDs:
7132 (Human); 21937 (Mouse); 25625 (Rat); 102135285 (Cynomolgus Monkey)
Alternate Names:
CD120a antigen; CD120a; FPF; p55; p55-R; p60; TNF RI; TNFARMGC19588; TNF-R; TNF-R1; TNFR1TBP1; TNFR55; TNF-R55; TNFR60; TNFRI; TNF-RI; TNF-R-I; TNFR-I; TNFRSF1A; tumor necrosis factor binding protein 1; Tumor necrosis factor receptor 1; tumor necrosis factor receptor 1A isoform beta; tumor necrosis factor receptor superfamily member 1A; tumor necrosis factor receptor superfamily, member 1A; tumor necrosis factor receptor type 1; Tumor necrosis factor receptor type I; tumor necrosis factor-alpha receptor

Assay Procedure

These assays utilize an anti-tag coated microplate. Standards, samples, and controls are added to plate, followed by subsequent addition of an antibody cocktail. Following a 1 hour incubation, plates are washed and substrate is added and incubated for 20 minutes. Stop solution is then added and plates are read using a standard microplate reader.

QuicKit ELISA assay procedure

FAQs

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