Human TNF-α ELISA Kit

Protocol Booklet

Key features and details

Specification: 96 Test
Sensitivity: 0.21 pg/ml (50 μl) ；0.26 pg/ml (10 μl)
Standard Curve Range: 1.37~1000 pg/ml
Standard Curve Gradient: 7 Points/3 Folds
Number of Incubations: 2
Sample Volume: 50 μl/10 μl
Assay type: Sandwich Elisa
Operation Duration: 120min

Brand:

CAT.NO. : AEH0002

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Size:

96T

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Product Details

Background

Tumor necrosis factor alpha (TNF-alpha ), also known as cachectin and TNFSF1A, is the prototypic ligand of the TNF superfamily. It is a pleiotropic molecule that plays a central role in inflammation, immune system development, apoptosis, and lipid metabolism. 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. Mouse 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 179 aa extracellular domain (ECD). Within the ECD, mouse TNF-alpha shares 95% aa identity with rat, and 80% aa identity with canine, equine, feline, human, rabbit, and porcine 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. 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. Shedding of membrane bound TNF-alpha by TACE/ADAM17 releases the bioactive cytokine, a 55 kDa soluble trimer containing the TNF-alpha extracellular domain. TNF-alpha binds the ubiquitous 55-60 kDa TNF RI and the hematopoietic cell-restricted 78-80 kDa TNF RII, both of which are also expressed as homotrimers. Both type I and type II receptors bind TNF-alpha with comparable affinity and can promote NFkB activation. Only TNF RI, however, contains a cytoplasmic death domain which triggers the activation of apoptosis. Soluble forms of both types of receptors are released into human serum and urine, and can neutralize the biological activity of TNF.

Typical data

pg/ml	O.D.		Average	Corrected
0	0.039	0.0402	0.0396
1.37	0.0588	0.0628	0.0608	0.0212
4.12	0.0819	0.0885	0.0852	0.0456
12.35	0.1693	0.1712	0.1703	0.1307
37.04	0.3876	0.3956	0.3916	0.352
111.11	0.9071	0.9969	0.952	0.9124
333.33	2.4084	2.3694	2.3889	2.3493
1000	4.3651	4.1387	4.2519	4.2123

Precision

	Intra-assay Precision			Inter-assay Precision
Sample Number	S1	S2	S3	S1	S2	S3
Sample Number	22	22	22	6	6	6
Average(pg/ml)	13	74.8	215.6	15.3	73.9	323.3
Standard Deviation	0.5	3.1	7.3	0.6	2.8	12.9
Coefficient of Variation(%)	3.7	4.1	3.4	4	3.8	4

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 six times on one plate to assess intra-assay precision.

Spike Recovery

The spike recovery was evaluated by spiking 3 levels of human TNF alpha into health human serum sample. The un-spiked serum was used as blank in this experiment.

The recovery ranged from 95% to 105% with an overall mean recovery of 99%.

Sample Values

Sample Matrix	Sample Evaluated	Range (pg/ml)	Detectable (%)	Mean of Detectable (pg/ml)
Serum	30	0.63-3.85	100	2.08

Serum/Plasma – Thirty samples from apparently healthy volunteers were evaluated for the presence of IL-10 in this assay. No medical histories were available for the donors.

n.d. = non-detectable. Samples measured below the sensitivity are considered to be non-detectable.

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