Catalog No. CSB-E07348r (96 T)       

 

l This immunoassay kit allows for the in vitro quantitative determination of rat VEGFR-2/Flk-1 concentrations in serum, plasma.

l Expiration date   six months from the date of manufacture

l FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

 

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Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell mitogen, which stimulates angiogenesis by binding to specific receptors (VEGFRs) on the surface of vascular endothelial cells. The VEGF liga-n-d/receptor signaling system plays a key role in vascular development a-n-d regulation of vascular permeability. All the three receptors for VEGF, VEGFR1~3, belong to CSF-1/PDGF receptor subfamily of receptor tyrosine kinase superfamily (RTKs), a-n-d contains seven Ig-like C2-type domains in the ECD, as well as a intracellular protein kinase domain. VEGFR2, also called as KDR or Flk-1, is identified as the receptor for VEGF a-n-d VEGFC a-n-d an early marker for endothelial cell progenitors, whose expression is restricted to endothelial cells in vivo. The adaptor protein SHB has been shown to interact with VEGFR2 in receptor tyrosine kinase signaling. In addition, VEGFR2 is able to interact with HIV-1 extracellular Tat protein upon VEGF activation, a-n-d seems to enhance angiogenesis in Kaposi's sarcoma lesions.

PRINCIPLE OF THE ASSAY The microtiter plate provided in this kit has been pre-coated with an antibody specific to VEGFR-2/Flk-1. Sta-n-dards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for VEGFR-2/Flk-1 a-n-d Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well a-n-d incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain VEGFR-2/Flk-1, biotin-conjugated antibody a-n-d enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution a-n-d the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of VEGFR-2/Flk-1 in the samples is then determined by comparing the O.D. of the samples to the sta-n-dard curve. DETECTION RANGE 0.31 ng/ml-20 ng/ml. The sta-n-dard curve concentrations used for the ELISA’s were 20 ng/ml, 10 ng/ml, 5 ng/ml, 2.5 ng/ml, 1.25 ng/ml, 0.63 ng/ml, 0.31 ng/ml.

SPECIFICITY This assay recognizes recombinant a-n-d natural rat VEGFR-2/Flk-1. No significant cross-reactivity or interference was observed. SENSITIVITY The minimum detectable dose of rat VEGFR-2/Flk-1 is typically less than 0.08 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. MATERIALS PROVIDED

1. Unopened test kits should be stored at 2-8?C upon receipt a-n-d the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date.

2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.  

3. A microtiter plate reader with a ba-n-dwidth of 10 nm or less a-n-d an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

 

1. Wash Buffer  If crystals have formed in the concentrate, warm up to room temperature a-n-d mix gently until the crystals have compley dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer.

 

 

2. Sta-n-dard  Centrifuge the sta-n-dard vial at 6000-10000rpm for 30s. Reconstitute the Sta-n-dard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 20 ng/ml. Allow the sta-n-dard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted sta-n-dard serves as the high sta-n-dard (20 ng/ml). The Sample Diluent serves as the zero sta-n-dard (0 ng/ml). Prepare fresh for each assay. Use within 4 hours a-n-d discard after use.

3. Biotin-antibody  Centrifuge the vial before opening. Dilute to the working concentration using Biotin-antibody Diluent(1:100),respectively. The suggested 100-fold dilution can be achieved by adding 10 uL sample to 990uL of Biotin-antibody Diluent for 1ml working solution. 

4. HRP-avidin  Centrifuge the vial before opening. Dilute to the working concentration using HRP-avidin Diluent(1:100), respectively. The suggested 100-fold dilution can be achieved by adding 10 uL sample to 990uL of HRP-avidin Diluent for 1ml working solution.

 

? Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.

? Pipettes a-n-d pipette tips.

? Deionized or distilled water.

? Squirt bottle, manifold dispenser, or automated microplate washer.

 

l Serum Use a serum separator tube (SST) a-n-d allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum a-n-d assay immediay or aliquot a-n-d store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.

l Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediay or aliquot a-n-d store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.

 

1. Add 100μl of Sta-n-dard, Blank, or Sample per well. Cover with the adhesive strip. Incubate for 2 hours at 37° C. 

2. Remove the liquid of each well, don’t wash. 

3. Add 100μl of Biotin-antibody working solution to each well. Incubate for 1 hour at 37°C. Biotin-antibody working solution may appear cloudy. Warm up to room temperature a-n-d mix gently until solution appears uniform.

4. Aspirate each well a-n-d wash, repeating the process three times for a total of three washes. Wash: Fill each well with Wash Buffer (200μl) a-n-d let it sta-n-d for 2 minutes, then remove the liquid by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel. Complete removal of liquid at each step is essential to good performance.

5. Add 100μl of HRP-avidin working solution to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C.

 

 

6. Repeat the aspiration a-n-d wash five times as step 4.

7. Add 90μl of TMB Substrate to each well. Incubate for 10-30 minutes at 37°C. Keeping the plate away from drafts a-n-d other temperature fluctuations in the dark. 

8. Add 50μl of Stop Solution to each well when the first four wells containing the highest concentration of sta-n-dards develop obvious blue color. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.

9. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.

 

Average the duplicate readings for each sta-n-dard, control, a-n-d sample a-n-d subtract the average zero sta-n-dard optical density. Create a sta-n-dard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a sta-n-dard curve by plotting

the mean absorbance for each sta-n-dard on the y-axis against the concentration on the x-axis a-n-d draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the VEGFR-2/Flk-1 concentrations versus the log of the O.D. a-n-d 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 sta-n-dard curve must be multiplied by the dilution factor. LIMITATIONS OF THE PROCEDURE

? The kit should not be used beyond the expiration date on the kit label.

? Do not mix or substitute reagents with those from other lots or sources.

? It is important that the Calibrator Diluent selected for the sta-n-dard curve be consistent with the samples being assayed.

? If samples generate values higher than the highest sta-n-dard, dilute the samples with the appropriate Calibrator Diluent a-n-d repeat the assay.

 

 

? Any variation in Sta-n-dard Diluent, operator, pipetting technique, washing technique, incubation time or temperature, a-n-d kit age can cause variation in binding.

? This assay is designed to eliminate interference by soluble receptors, binding proteins, a-n-d other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

 

? Centrifuge vials before opening to collect contents.

? When mixing or reconstituting protein solutions, always avoid foaming.

? To avoid cross-contamination, change pipette tips between additions of each sta-n-dard level, between sample additions, a-n-d between reagent additions. Also, use separate reservoirs for each reagent.

? When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, a-n-d/or rotating the plate 180 degrees between wash steps may improve assay precision.

 

 

? To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

? Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue.

? Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.