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  • 產品名稱：Osteopontin ELISA Kit(Secretedphosphoprotein1)

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  • 產品廠商：國內供應3
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Osteopontin ELISA Kit(Secretedphosphoprotein1)
詳情介紹：
Purpose This immunoassay kit allows for the specific measurement of Rat Osteopontin (OPN) concentrations in cell culture supernates, serum and plasma.
Sample Type Cell Culture Supernatant, Serum, Plasma
Analytical Method Quantitative
Detection Method Colorimetric
Specificity This assay recognizes recombinant and natural Rat Osteopontin.
Cross-Reactivity (Details) No significant cross-reactivity or interference was observed.
Characteristics Rattus norvegicus,Rat,Osteopontin,Bone sialoprotein 1,Secreted phosphoprotein 1,SPP-1,Spp1,2b7,Spp-1
Components Reagent (Quantity ): Assay plate (1), Standard (2), Sample Diluent (1 × 20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1 × 120μl), Detection Reagent B (1 × 120μl), Wash Buffer (25 x concentrate) (1 × 30ml), Substrate (1x10ml), Stop Solution (1 x 10ml)
Alternative Name Spp1 (SPP1 ELISA Kit Abstract)
Background Osteopontin (OPN), also known as early T lymphocyte activation 1 (Eta-1), is a secreted multifunctional glyco-phosphoprotein with roles in bone metabolism, immune regulation, tissue remodeling, cell survival, and tumor progression . Gene structure and chromosomal location identify OPN as a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family that also includes bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein(DSPP), enamelin (ENAM), and matrix extracellular phosphoglycoprotein (MEPE). Murine OPN issynthesized as a 294 amino acid (aa) precursor protein with a predicted 16 aa signal peptide and a highly unusual mature protein sequence, containing 68 acidic aa and 23 potential Ser/Thr phosphorylation sites. Although the predicted molecular weight of OPN is 31 kDa,phosphorylation and N- and O-glycosylation may allow it to appear as large as 75 kDa. Variability in post-translational modifications can influence the activity of OPN. Osteopontin, as indicated by its name meaning “bone bridging”, is highly expressed in mineralized tissues. It is also expressed in other tissues including cartilage, kidney, vascular tissues, activated macrophages, lymphocytes, and epithelia. A portion of cell expressed OPN is also retained intracellularly. In vitro, OPN stimulates the adhesion of osteoclasts to bone, and bone resorption is blocked by inhibition of this interaction. Knockout mice have outwardly normal bone development, but exhibit deficient postnatal bone resorption in several contexts, supporting a role for OPN in osteoclast function. In kidney epithelia, OPN is upregulated by high concentrations of oxalate and inhibits calcium oxalate crystal nucleation and growth. In endothelial and smooth muscle cells, OPN is upregulated by high phosphate concentration and during atherosclerosis, binding of OPN to hydroxyapatite inhibits calcification of blood vessels and heart valves. OPN expression by macrophages and T cells is upregulated by inflammatory mediators including LPS,NO, IL-1beta, and TNF-alpha. OPN regulates macrophage differentiation and recruitment. It also functions as a chemotactic factor and co-stimulator of T cells and may act as a Th1 cytokine, stimulating IL-12 production. OPN production by macrophages is upregulated at sites of tissue remodeling including the placenta, endometrium and myocardium post-infarction.
Gene ID 2981
Pathways Regulation of Cell Size
Sample Volume 100 μL
Plate Pre-coated
Protocol This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal 2 antibody specific for Osteopontin has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Osteopontin present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for Osteopontin is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of Osteopontin bound in the initial step. The color development is stopped and the intensity of the color is measured.
Reagent Preparation

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 2000 pg/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (2000 pg/mL). The Sample Diluent serves as the zero standard (0 pg/mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.
Sample Collection Cell culture supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay.
Assay Procedure

Allow all reagents to reach room temperature. Arrange and label required number of strips.
1. Prepare all reagents, working standards and samples as directed in the previous sections.
2. Add 100 uL of Standard, Control, or sample* per well. Cover with the adhesive strip. Incubate for 2 hours at 37 °C.
3. Remove the liquid of each well, don’t wash.
4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
7. Repeat the aspiration/wash as in step
5. 8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Important Note:1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. 4
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
3. Duplication of all standards and specimens, although not required, is recommended.
4. When mixing or reconstituting protein solutions, always avoid foaming.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
Calculation of Results

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the Osteopontin concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Restrictions For Research Use only
Handling Advice 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
3.
Storage 4 °C/-20 °C
Storage Comment The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.