Human TNF RI/TNFRSF1A Quantikine ELISA Kit
R&D Systems, part of Bio-Techne | Catalog # DRT100
Key Product Details
Assay Length
4.5 hours
Sample Type & Volume Required Per Well
Cell Culture Supernates (200 µL), Serum (20 µL), EDTA Plasma (20 µL), Heparin Plasma (20 µL), Citrate Plasma (20 µL), Urine (20 µL)
Sensitivity
1.2 pg/mL
Assay Range
7.8-500 pg/mL (Cell Culture Supernates, Serum, EDTA Plasma, Heparin Plasma, Citrate Plasma, Urine)
Product Summary for Human TNF RI/TNFRSF1A Quantikine ELISA Kit
The Quantikine Human sTNF RI Immunoassay is a 4.5 hour solid phase ELISA designed to measure sTNF RI in cell culture supernates, serum, plasma, and urine. It contains E. coli-expressed recombinant human sTNF RI, as well as antibodies raised against this polypeptide. The recombinant protein represents the non-glycosylated, N-terminal methionyl form of the naturally occurring human soluble Type I receptor for TNF with an apparent molecular weight of approximately 18.6 kDa. This immunoassay has been shown to accurately quantitate the recombinant sTNF RI. Results obtained on samples containing natural sTNF RI showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values of natural sTNF RI. Since the measurement of human sTNF RI by this immunoassay is relatively insensitive to added TNF-alpha or TNF-beta, it is probable that this measurement corresponds to the total amount of the soluble receptor present in samples (i.e. the total amount of free receptor plus the total amount of receptor bound to TNF).
Product Specifications
Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Measurement
Quantitative ELISA
Detection Method
Colorimetric - 450nm (TMB)
Conjugate
HRP
Reactivity
Human
Specificity
Natural and recombinant human sTNF RI
Cross-reactivity
< 0.5% cross-reactivity observed with available related molecules. < 50% cross-species reactivity observed with species tested.
Interference
Interference observed with 1 or more available related molecules.
Sample Values
Serum/Plasma/Urine - Samples from apparently healthy volunteers were evaluated for the
presence of human TNF RI in this assay. No medical histories were available for the donors used
in this study.
*Values are actual and not normalized for creatinine content.
| Sample Type | Mean (pg/mL) | Range (pg/mL) | Standard Deviation (pg/mL) |
| Serum (n=40) | 1198 | 749-1966 | 256 |
| EDTA plasma (n=40) | 914 | 484-1407 | 208 |
| Heparin plasma (n=40) | 1015 | 512-1739 | 245 |
| Citrate plasma (n=40) | 856 | 488-1598 | 210 |
| Urine* (n=33) | 1029 | 173-4030 | 832 |
Cell Culture Supernates - Human peripheral blood mononuclear cells (1 x 106
cells/mL)
were cultured in RPMI supplemented with 10% fetal bovine serum, 50 μM beta-mercaptoethanol,
2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. Cells were
stimulated with 10 μg/mL PHA, 10 μg/mL PHA + 10 ng/mL recombinant human (rh) IL-2,
50 ng/mL PMA, or 50 ng/mL LPS. Aliquots of the cell culture supernates were removed on days
1, 3, and 5 and assayed for levels of human TNF RI.
| Stimulant | Day 1 (pg/mL) | Day 3 (pg/mL) | Day 5 (pg/mL) |
| PHA | 24 | 76 | 141 |
| PHA + rhIL-2 | 26 | 71 | 143 |
| PMA | 16 | 27 | 67 |
| LPS | 17 | 36 | 49 |
Precision
Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision.
Cell Culture Supernates, Urine
| Intra-Assay Precision | Inter-Assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) | 41.5 | 122 | 231 | 54.0 | 251 | 350 |
| Standard Deviation | 2.14 | 5.38 | 11.0 | 2.7 | 9.7 | 23.3 |
| CV% | 5.2 | 4.4 | 4.8 | 5.0 | 3.9 | 6.7 |
Citrate Plasma, EDTA Plasma, Heparin Plasma, Serum
| Intra-Assay Precision | Inter-Assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) | 69.0 | 198 | 355 | 54.8 | 252 | 356 |
| Standard Deviation | 3.24 | 7.17 | 17.8 | 4.8 | 9.3 | 20.6 |
| CV% | 4.7 | 3.6 | 5.0 | 8.8 | 3.7 | 5.8 |
Recovery for Human TNF RI/TNFRSF1A Quantikine ELISA Kit
The recovery of sTNF RI spiked to three levels in samples throughout the range of the assay in various matrices was evaluated.
| Sample Type | Average % Recovery | Range % |
|---|---|---|
| Cell Culture Media (n=8) | 85 | 80-92 |
| EDTA Plasma (n=8) | 86 | 70-97 |
| Heparin Plasma (n=8) | 93 | 79-103 |
| Serum (n=8) | 90 | 77-103 |
| Urine (n=8) | 85 | 71-109 |
Linearity
To assess the linearity of the assay, samples containing and/or spiked with high concentrations of sTNF RI were serially diluted with the appropriate Calibrator Diluent to produce samples with values within the dynamic range of the assay.
Scientific Data Images for Human TNF RI/TNFRSF1A Quantikine ELISA Kit
Human TNF RI/TNFRSF1A ELISA Calibrator Diluent RD5-5 Standard Curve
Human TNF RI/TNFRSF1A ELISA Calibrator Diluent RD6O Standard Curve
Preparation and Storage
Shipping
The product is shipped at ambient temperature. 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 RI/TNFRSF1A
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
Alternate Names
CD120a, TNFRI, TNFRSF1A
Gene Symbol
TNFRSF1A
Additional TNF RI/TNFRSF1A Products
Product Documents for Human TNF RI/TNFRSF1A Quantikine ELISA Kit
Product Specific Notices for Human TNF RI/TNFRSF1A Quantikine ELISA Kit
For research use only
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⚠ WARNING: This product can expose you to chemicals including N,N-Dimethylforamide, which is known to the State of California to cause cancer. For more information, go to www.P65Warnings.ca.gov.