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Human TNF-alpha ELISA Kit - Quantikine Best Seller

Catalog # DTA00D | R&D Systems, Inc. a Bio-Techne Brand
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Key Product Details

Assay Length

80 minutes

Sample Type & Volume Required Per Well

Cell Culture Supernates (50 µL), Serum (50 µL), Plasma (50 µL)

Sensitivity

6.23 pg/mL

Assay Range

15.6-1000 pg/mL (Cell Culture Supernates, Serum, Plasma)

Product Summary for Human TNF-alpha Quantikine ELISA Kit

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 - Thirty 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, 15.6 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 ng/mL of PMA and 500 ng/mL of Ionomycin Calcium Salt (Tocris®, Catalog # 1704) for 24 hours. Aliquots of the culture supernates were removed, assayed for levels of human TNF-alpha, and measured 1355 pg/mL and 15,240 pg/mL, respectively.

CD4+ T cells were isolated from human PBMCs using the MagCellect™ Human CD4+ T cell Isolation Kit (R&D Systems, Catalog # MAGH102) before being differentiated into Th1 cells for 5 days and then treated with 10 ng/mL of PMA and 500 ng/mL of Ionomycin for an additional 24 hours. An aliquot of the cell culture supernate was removed, assayed for human TNF-alpha, and measured 81,100 pg/mL.

Human monocyte-derived macrophages (MDM) were obtained from PBMCs and attached monocytes were cultured in RPMI supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, and 100 ng/mL of recombinant human M-CSF for 5 days. Cells were treated with 1 μg/mL LPS and 40 ng/mL of recombinant human IFN-gamma overnight. An aliquot of the cell culture supernate was removed, assayed for human TNF-alpha, and measured 8550 pg/mL. 

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, 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) 155 304 606 155 298 592
Standard Deviation 4.08 6.62 18.3 11.3 21.8 49.9
CV% 2.6 2.2 3.0 7.3 7.3 8.4

Recovery for Human TNF-alpha Quantikine ELISA Kit

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) 103 98-110
EDTA Plasma (n=4) 90 75-101
Heparin Plasma (n=4) 90 82-95
Serum (n=4) 88 79-98

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.

Human TNF-alpha ELISA Linearity

Scientific Data Images for Human TNF-alpha Quantikine ELISA Kit

Human TNF-alpha ELISA Standard Curve

Human TNF-alpha ELISA 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-alpha

Tumor Necrosis Factor alpha (TNF-alpha), also known as cachectin and TNFSF1A, is the prototypic ligand of the TNF superfamily (1). It is a pleiotropic molecule that plays a central role in inflammation, immune system development, apoptosis, and lipid metabolism (2-5). TNF-alpha is also involved in a number of pathological conditions including asthma, Crohn's disease, rheumatoid arthritis, neuropathic pain, obesity, type 2 diabetes, septic shock, autoimmunity, and cancer (5-11).  
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

7124 (Human); 21926 (Mouse); 24835 (Rat); 397086 (Porcine); 280943 (Bovine); 403922 (Canine); 102139631 (Cynomolgus Monkey); 100033834 (Equine); 493755 (Feline); 100009088 (Rabbit)

Gene Symbol

TNF

Additional TNF-alpha Products

Product Documents for Human TNF-alpha Quantikine ELISA Kit

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

Product Specific Notices for Human TNF-alpha Quantikine ELISA Kit

For research use only

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