DESCRIPTION

TNF, as also known as TNF-alpha, or cachectin, is a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. It is expressed as a 26 kDa membrane bound protein and is then cleaved by TNF-alpha converting enzyme (TACE) to release the soluble 17 kDa monomer, which forms homotrimers in circulation. It is produced chiefly by activated macrophages, although it can be produced by many other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons. It can bind to, and thus functions through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. This cytokine is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. Dysregulation of TNF production has been implicated in a variety of human diseases including Alzheimer's disease, cancer, major depression and inflammatory bowel disease (IBD).

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with HRP) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with HRP also binds to the analyte. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns solution yellow. The color intensity is proportional to the quantity of bound protein which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	20	130.2	4.7	3.6
2	20	27.6	1.3	4.9
3	20	5.6	0.5	9.3

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	24	124.7	5.3	4.2
2	24	29.2	1.5	5.0
3	24	6.5	0.6	9.5

RECOVERY

The recovery of human TNF-alpha spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:2	85	79-98
Cell culture supernatant	1:32	88	84-91
	1:64	92	87-95

LINEARITY

To assess the linearity of the assay, human plasma samples were spiked with high concentrations of human TNF-alpha and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Cell culture supernatant samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The cell culture supernatant was initially diluted 1:8.)

		Human plasma(Sample Diluent PT 1)	Cell culture supernatant(Sample Diluent PT 4B1)
1:2	Average% of Expected	73	100
	Range (%)	72-74	-
1:4	Average% of Expected	92	107
	Range (%)	91-94	104-109
1:8	Average% of Expected	106	107
	Range (%)	105-107	106-108
1:16	Average% of Expected	81	114
	Range (%)	74-88	113-114

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human TNF-alpha produced at Sunlong Medical™.