Human TNFSF4/OX40L ELISA Kit (HUFI00274)- High Sensitivity

Product Name:	Human TNFSF4/OX40L ELISA Kit
Product Code:	HUFI00274
Size:	96 Assays
Alias:	OX40L, TNFSF4, CD252, gp34, CD134 ligand, CD134L, CD252 antigen, Glycoprotein Gp34, GP34, OX40 antigen ligand, OX40 ligand, OX-40L, OX4OL, TXGP1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human TNFSF4/OX40L concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human TNFSF4/OX40L and the recovery rates were calculated by comparing the measured value to the expected amount of Human TNFSF4/OX40L in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	91-101	97
EDTA plasma(n=5)	92-105	96
UFH plasma(n=5)	85-98	91

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TNFSF4/OX40L and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	97-105%	86-104%	89-103%
EDTA plasma(n=5)	85-101%	85-98%	86-100%
UFH plasma(n=5)	81-97%	81-99%	88-99%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P23510

UniProt Protein Function:	TNFSF4: Cytokine that binds to TNFRSF4. Co-stimulates T-cell proliferation and cytokine production. Genetic variations in TNFSF4 influence susceptibility to systemic lupus erythematosus (SLE). SLE is a chronic, inflammatory and often febrile multisystemic disorder of connective tissue. It affects principally the skin, joints, kidneys and serosal membranes. It is thought to represent a failure of the regulatory mechanisms of the autoimmune system. The upstream region of TNFSF4 contains a single risk haplotype for SLE, which is correlated with increased expression of both cell-surface TNFSF4 and TNFSF4 transcripts. Increased levels of TNFSF4 are thought to augment T-cell-APC interaction and the functional consequences of T-cell activation, thereby destabilizing peripheral tolerance. Belongs to the tumor necrosis factor family.
UniProt Protein Details:	Protein type:Membrane protein, integral; Cell cycle regulation; Cytokine Chromosomal Location of Human Ortholog: 1q25 Cellular Component: extracellular space; cell surface; integral to plasma membrane Molecular Function:cytokine activity; tumor necrosis factor receptor superfamily binding; tumor necrosis factor receptor binding; receptor binding Biological Process: negative regulation of T-helper 1 cell differentiation; positive regulation of interleukin-12 production; negative regulation of cytokine secretion; positive regulation of T cell cytokine production; defense response to nematode; signal transduction; positive regulation of activated T cell proliferation; positive regulation of interleukin-10 production; positive regulation of interleukin-4 production; T cell proliferation; regulation of adaptive immune response; positive regulation of T-helper 2 cell differentiation; negative regulation of regulatory T cell differentiation; negative regulation of interferon-gamma production; positive regulation of cell proliferation; positive regulation of interleukin-13 production; positive regulation of memory T cell differentiation; cholesterol metabolic process; positive regulation of T-helper 2 type immune response; response to virus; negative regulation of transcription factor activity; positive regulation of immunoglobulin mediated immune response; positive regulation of interleukin-6 production; positive regulation of CD4-positive, alpha beta T cell differentiation; positive regulation of immunoglobulin secretion; positive regulation of interferon-gamma production; negative regulation of interleukin-17 production; positive regulation of B cell activation; regulation of inflammatory response; positive regulation of interleukin-2 production; immune response; positive regulation of alpha-beta T cell proliferation; negative regulation of transcription, DNA-dependent; acute inflammatory response; positive regulation of inflammatory response Disease: Myocardial Infarction, Susceptibility To
NCBI Summary:	This gene encodes a cytokine of the tumor necrosis factor (TNF) ligand family. The encoded protein functions in T cell antigen-presenting cell (APC) interactions and mediates adhesion of activated T cells to endothelial cells. Polymorphisms in this gene have been associated with Sjogren's syndrome and systemic lupus erythematosus. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jul 2014]
UniProt Code:	P23510
NCBI GenInfo Identifier:	121540
NCBI Gene ID:	7292
NCBI Accession:	P23510.1
UniProt Secondary Accession:	P23510,Q5JZA5, Q8IV74, Q9HCN9,
UniProt Related Accession:	P23510
Molecular Weight:	15,427 Da
NCBI Full Name:	Tumor necrosis factor ligand superfamily member 4
NCBI Synonym Full Names:	tumor necrosis factor (ligand) superfamily, member 4
NCBI Official Symbol:	TNFSF4
NCBI Official Synonym Symbols:	GP34; CD252; OX4OL; TXGP1; CD134L; OX-40L
NCBI Protein Information:	tumor necrosis factor ligand superfamily member 4; CD134 ligand; glycoprotein Gp34; OX40 antigen ligand; tax-transcriptionally activated glycoprotein 1 (34kD)
UniProt Protein Name:	Tumor necrosis factor ligand superfamily member 4
UniProt Synonym Protein Names:	Glycoprotein Gp34; OX40 ligand; OX40L; TAX transcriptionally-activated glycoprotein 1; CD_antigen: CD252
UniProt Gene Name:	TNFSF4
UniProt Entry Name:	TNFL4_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Human TNFSF4/OX40L ELISA Kit (HUFI00274)

Description