Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	7.81-500 pg/mL
Sensitivity:	4.69 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human TNF- alpha in samples. No significant cross-reactivity or interference between Human TNF- alpha and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human TNF- alpha. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human TNF- alpha and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human TNF- alpha, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human TNF- alpha. The concentration of Human TNF- alpha in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	TNF-a: Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation. Homotrimer. Interacts with SPPL2B. Belongs to the tumor necrosis factor family.
UniProt Protein Details:	Protein type:Membrane protein, integral; Motility/polarity/chemotaxis; Apoptosis; Cytokine Chromosomal Location of Human Ortholog: 6p21. 3 Cellular Component: extracellular space; recycling endosome; cell surface; integral to plasma membrane; extracellular region; plasma membrane; external side of plasma membrane; phagocytic cup; lipid raft Molecular Function:identical protein binding; protein binding; protease binding; cytokine activity; tumor necrosis factor receptor binding Biological Process: positive regulation of JNK activity; extracellular matrix organization and biogenesis; positive regulation of nitric oxide biosynthetic process; positive regulation of NFAT protein import into nucleus; activation of MAPK activity; positive regulation of osteoclast differentiation; positive regulation of apoptosis; positive regulation of transcription, DNA-dependent; response to glucocorticoid stimulus; positive regulation of caspase activity; positive regulation of NF-kappaB import into nucleus; osteoclast differentiation; positive regulation of translational initiation by iron; positive regulation of membrane protein ectodomain proteolysis; activation of NF-kappaB transcription factor; positive regulation of MAP kinase activity; tumor necrosis factor-mediated signaling pathway; positive regulation of phagocytosis; negative regulation of interleukin-6 production; JNK cascade; negative regulation of osteoblast differentiation; positive regulation of action potential; regulation of immunoglobulin secretion; embryonic gut development; negative regulation of protein complex disassembly; positive regulation of cytokine production; response to drug; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of heterotypic cell-cell adhesion; positive regulation of mitosis; response to virus; positive regulation of interleukin-8 biosynthetic process; glucose metabolic process; positive regulation of interleukin-6 production; negative regulation of fat cell differentiation; positive regulation of chemokine production; negative regulation of cytokine secretion during immune response; positive regulation of protein transport; detection of mechanical stimulus involved in sensory perception of pain; cell activation; defense response to Gram-positive bacterium; induction of apoptosis via death domain receptors; DNA damage response, signal transduction resulting in induction of apoptosis; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription factor activity; negative regulation of L-glutamate transport; response to activity; negative regulation of transcription, DNA-dependent; skeletal muscle contraction; sequestering of triacylglycerol; positive regulation of smooth muscle cell proliferation; negative regulation of transcription from RNA polymerase II promoter; positive regulation of interleukin-18 production; chronic inflammatory response to antigenic stimulus; response to salt stress; positive regulation of synaptic transmission; positive regulation of hair follicle development; negative regulation of cell proliferation; response to radiation; negative regulation of lipid catabolic process; positive regulation of neuron apoptosis; protein kinase B signaling cascade; lipopolysaccharide-mediated signaling pathway; positive regulation of chronic inflammatory response to antigenic stimulus; inflammatory response; regulation of I-kappaB kinase/NF-kappaB cascade; caspase activation; positive regulation of humoral immune response mediated by circulating immunoglobulin; positive regulation of protein complex disassembly; transformed cell apoptosis; MAPKKK cascade; calcium-mediated signaling; positive regulation of peptidyl-serine phosphorylation; humoral immune response; positive regulation of protein kinase B signaling cascade; positive regulation of interferon-gamma production; negative regulation of glucose import; positive regulation of programmed cell death; positive regulation of chemokine biosynthetic process; positive regulation of protein complex assembly; negative regulation of viral genome replication; protein import into nucleus, translocation; positive regulation of protein kinase activity; response to hypoxia; positive regulation of fever; activation of MAPKKK activity; receptor biosynthetic process; positive regulation of protein amino acid phosphorylation; leukocyte tethering or rolling; negative regulation of myoblast differentiation; regulation of insulin secretion; positive regulation of cytokine secretion Disease: Asthma, Susceptibility To; Migraine With Or Without Aura, Susceptibility To, 1; Malaria, Susceptibility To
NCBI Summary:	This gene encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. This cytokine is mainly secreted by macrophages. 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. This cytokine has been implicated in a variety of diseases, including autoimmune diseases, insulin resistance, and cancer. Knockout studies in mice also suggested the neuroprotective function of this cytokine. [provided by RefSeq, Jul 2008]
UniProt Code:	P01375
NCBI GenInfo Identifier:	135934
NCBI Gene ID:	7124
NCBI Accession:	P01375. 1
UniProt Secondary Accession:	P01375,O43647, Q9P1Q2, Q9UIV3,
UniProt Related Accession:	P01375
Molecular Weight:	25,644 Da
NCBI Full Name:	Tumor necrosis factor
NCBI Synonym Full Names:	tumor necrosis factor
NCBI Official Symbol:	TNF
NCBI Official Synonym Symbols:	DIF; TNFA; TNFSF2; TNF-alpha
NCBI Protein Information:	tumor necrosis factor; TNF-a; cachectin; APC1 protein; TNF, monocyte-derived; TNF, macrophage-derived; tumor necrosis factor-alpha; tumor necrosis factor ligand superfamily member 2
UniProt Protein Name:	Tumor necrosis factor
UniProt Synonym Protein Names:	Cachectin; TNF-alpha; Tumor necrosis factor ligand superfamily member 2; TNF-a
Protein Family:	Tumor necrosis factor
UniProt Gene Name:	TNF
UniProt Entry Name:	TNFA_HUMAN

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (pg/mL)	RLU	Average	Corrected
500	49282 53792	51537	51508
250	19118 22878	20998	20969
125	9582 9210	9396	9367
62.5	4339 4685	4512	4483
31.25	2452 2146	2299	2270
15.63	1341 1159	1250	1221
7.81	735 745	740	711
0	29 29	29	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human TNF- alpha were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human TNF- alpha were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/mL)	25.60	58.03	169.01	27.58	54.45	158.50
Standard deviation	2.69	5.50	13.54	2.46	6.40	14.99
C V (%)	10.51	9.48	8.01	8.92	11.75	9.46

Recovery

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

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	100-113	107
EDTA plasma (n=5)	92-103	98
Cell culture media (n=5)	87-101	92

Linearity

Samples were spiked with high concentrations of Human TNF- alpha and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	86-99	98-112	97-110
1:2	Average (%)	93	105	103
1:4	Range (%)	99-111	101-117	85-98
1:4	Average (%)	105	109	90
1:8	Range (%)	90-102	91-105	93-106
1:8	Average (%)	97	99	98
1:16	Range (%)	103-121	85-96	103-116
1:16	Average (%)	110	91	109

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro CLIA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.