Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	15.63-1000 pg/mL
Sensitivity:	9.38 pg/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human TNF- beta in samples. No significant cross-reactivity or interference between Human TNF- beta and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human TNF- beta. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human TNF- beta 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- beta, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human TNF- beta. The concentration of Human TNF- beta in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	LTA: Cytokine that in its homotrimeric form binds to TNFRSF1A/TNFR1, TNFRSF1B/TNFBR and TNFRSF14/HVEM. In its heterotrimeric form with LTB binds to TNFRSF3/LTBR. Lymphotoxin is produced by lymphocytes and cytotoxic for a wide range of tumor cells in vitro and in vivo. Genetic variations in LTA are a cause of susceptibility psoriatic arthritis (PSORAS). PSORAS is an inflammatory, seronegative arthritis associated with psoriasis. It is a heterogeneous disorder ranging from a mild, non-destructive disease to a severe, progressive, erosive arthropathy. Five types of psoriatic arthritis have been defined: asymmetrical oligoarthritis characterized by primary involvement of the small joints of the fingers or toes; asymmetrical arthritis which involves the joints of the extremities; symmetrical polyarthritis characterized by a rheumatoidlike pattern that can involve hands, wrists, ankles, and feet; arthritis mutilans, which is a rare but deforming and destructive condition; arthritis of the sacroiliac joints and spine (psoriatic spondylitis). Belongs to the tumor necrosis factor family.
UniProt Protein Details:	Protein type:Cytokine; Apoptosis; Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 6p21. 3 Cellular Component: extracellular space; membrane Molecular Function:cytokine activity; tumor necrosis factor receptor binding; receptor binding Biological Process: response to drug; positive regulation of humoral immune response mediated by circulating immunoglobulin; apoptosis; positive regulation of apoptosis; response to lipopolysaccharide; signal transduction; lymph node development; humoral immune response; defense response to Gram-positive bacterium; positive regulation of interferon-gamma production; cell-cell signaling; negative regulation of fibroblast proliferation; response to hypoxia; positive regulation of chronic inflammatory response to antigenic stimulus; response to nutrient Disease: Psoriatic Arthritis, Susceptibility To; Myocardial Infarction, Susceptibility To; Leprosy, Susceptibility To, 4
NCBI Summary:	The encoded protein, a member of the tumor necrosis factor family, is a cytokine produced by lymphocytes. The protein is highly inducible, secreted, and forms heterotrimers with lymphotoxin-beta which anchor lymphotoxin-alpha to the cell surface. This protein also mediates a large variety of inflammatory, immunostimulatory, and antiviral responses, is involved in the formation of secondary lymphoid organs during development and plays a role in apoptosis. Genetic variations in this gene are associated with susceptibility to leprosy type 4, myocardial infarction, non-Hodgkin's lymphoma, and psoriatic arthritis. Alternatively spliced transcript variants have been observed for this gene. [provided by RefSeq, Jul 2012]
UniProt Code:	P01374
NCBI GenInfo Identifier:	135940
NCBI Gene ID:	4049
NCBI Accession:	P01374. 2
UniProt Secondary Accession:	P01374,Q8N4C3, Q9UKS8,
UniProt Related Accession:	P01374
Molecular Weight:	22,297 Da
NCBI Full Name:	Lymphotoxin-alpha
NCBI Synonym Full Names:	lymphotoxin alpha
NCBI Official Symbol:	LTA
NCBI Official Synonym Symbols:	LT; TNFB; TNFSF1
NCBI Protein Information:	lymphotoxin-alpha; LT-alpha; TNF-beta; TNF superfamily, member 1; tumor necrosis factor beta; tumor necrosis factor ligand superfamily member 1
UniProt Protein Name:	Lymphotoxin-alpha
UniProt Synonym Protein Names:	TNF-beta; Tumor necrosis factor ligand superfamily member 1
Protein Family:	Lymphotoxin
UniProt Gene Name:	LTA
UniProt Entry Name:	TNFB_HUMAN

As the OD 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)	O.D	Average	Corrected
1000	2.405 2.411	2.408	2.329
500	1.67 1.726	1.698	1.619
250	0.931 0.905	0.918	0.839
125	0.531 0.535	0.533	0.454
62.5	0.268 0.242	0.255	0.176
31.25	0.19 0.17	0.18	0.101
15.63	0.124 0.138	0.131	0.052
0	0.077 0.081	0.079	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human TNF- beta 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- beta 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)	50.20	90.80	409.30	52.00	96.10	426.70
Standard deviation	2.50	3.80	13.90	3.10	4.80	21.30
C V (%)	4.98	4.19	3.40	5.96	4.99	4.99

Recovery

The recovery of Human TNF- beta 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)	94-106	100
EDTA plasma (n=5)	94-109	101
Cell culture media (n=5)	85-100	92

Linearity

Samples were spiked with high concentrations of Human TNF- beta 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 (%)	94-106	99-112	90-105
1:2	Average (%)	99	105	96
1:4	Range (%)	96-107	83-97	94-112
1:4	Average (%)	101	88	102
1:8	Range (%)	98-112	83-94	90-105
1:8	Average (%)	104	89	98
1:16	Range (%)	97-113	84-97	93-109
1:16	Average (%)	103	89	101

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 ELISA 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	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
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 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.