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  • 產(chǎn)品名稱：VWF ELISA Kit(VonWillebrandFactor)

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  • 產(chǎn)品廠商：國內(nèi)供應(yīng)3
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VWF ELISA Kit(VonWillebrandFactor)
詳情介紹：
Purpose This immunoassay kit allows for the specific measurement of Porcine Von Willebrand Factor (vWF)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 porcine vWF.
Cross-Reactivity (Details) No significant cross-reactivity or interference was observed.
Characteristics Sus scrofa,Pig,von Willebrand factor,vWF,VWF,F8VWF
Components Reagent (Quantity ): Assay plate (1), Standard (2), Sample Diluent (1 × 20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent B (1 × 120μl), Detection Reagent B (1 × 120μl), Wash Buffer (25 x concentrate) (1 × 30ml), Substrate (1x10ml), Stop Solution (1 x 10ml)
Alternative Name VWF (VWF ELISA Kit Abstract)
Background Von Willebrand factor is a blood glycoprotein involved in coagulation. It is deficient or defective in von Willebrand disease and is involved in a large number of other diseases, including thrombotic thrombocytopenic purpura, Heyde's syndrome, and possibly hemolytic-uremic syndrome.vWF is a large multimeric glycoprotein present in blood plasma and produced constitutively in endothelium (in the Weibel-Palade bodies), megakaryocytes (alpha-granules of platelets), and subendothelial connective tissue.The basic vWF monomer is a 2050 amino acid protein. Von Willebrand factor is not an enzyme and therefore has no catalytic activity. Its primary function is binding to other proteins, particularly Factor VIII and it is important in platelet adhesion to wound sites. vWF binds to a number of cells and molecules. The most important ones are: Factor VIII is bound to vWF whilst inactive in circulation, vWF binds to collagen, e.g., when it is exposed in endothelial cells due to damage occurring to the blood vessel. vWF binds to platelet gpIb when it forms a complex with gpIX and gpV, this binding occurs under all circumstances, but is most efficient under high shear stress. vWF binds to other platelet receptors when they are activated, e.g., by thrombin (i.e., when coagulation has been stimulated). vWF appears to play a major role blood coagulation, and vWF deficiency or dysfunction (von Willebrand disease) therefore leads to a bleeding tendency, which is most apparent in tissues having high blood flow shear in narrow vessels. From studies it appears that vWF uncoils under these circumstances, decelerating passing platelets.
Gene ID 3976
Sample Volume 100 μL
Plate Pre-coated
Protocol This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for VWF has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any VWF present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for VWF 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 VWF 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 1000 ug/L. 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 (1000 ug/L). The Sample Diluent serves as the zero standard (0 ug/L). 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 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. 2.
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 3 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.
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 PAP 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.
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.