Shanghai Korain Biotech Co., Ltd
Shanghai Korain Biotech Co Ltd
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96 Wells Customized Mouse Vascular Endothelial Cell Growth Factor ELISA Kit
Cat.No E0114Mo
Standard Curve Range: 5ng/L - 1800ng/L
Sensitivity: 2.48ng/L
Size: 96 wells
Intended Use
This sandwich kit is for the accurate quantitative detection of Mouse Vascular Endothelial Cell Growth Factor (also known as VEGF) in serum, plasma, cell culture supernates, cell lysates, tissue homogenates.
Assay Principle
This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Mouse VEGF antibody. VEGF present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Mouse VEGF Antibody is added and binds to VEGF in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated VEGF antibody. After incubation unbound Streptavidin-HRP is washed away during a washing step. Substrate solution is then added and color develops in proportion to the amount of Mouse VEGF. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.
Precautions
Specimen Collection
Serum Allow serum to clot for 10-20 minutes at room temperature. Centrifuge at 2000-3000 RPM for 20 minutes.
Plasma Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 2000-3000 RPM at 2 - 8°C within 30 minutes of collection.
Urine Collect by sterile tube. Centrifuge at 2000-3000 RPM for approximately 20 minutes. When collecting pleuroperitoneal fluid and cerebrospinal fluid, please follow the procedures above-mentioned.
Cell Culture Supernatant Collect by sterile tubes when examining secrete components. Centrifuge at 2000-3000 RPM for approximately 20 minutes. Collect the supernatants carefully. When examining the components within the cell, use PBS (pH 7.2-7.4) to dilute cell suspension to the cell concentration of approximately 1 million/ml. Damage cells through repeated freeze-thaw cycles to let out the inside components. Centrifuge at 2000-3000 RPM for approximately 20 minutes.
Tissue Rinse tissues in PBS (pH 7.4) to remove excess blood thoroughly and weigh before homogenization. Mince tissues and homogenize them in PBS (pH7.4) with a glass homogenizer on ice. Thaw at 2-8°C or freeze at -20°C. Centrifuge at 2000-3000 RPM for approximately 20 minutes.
*Sample can't be diluted with this kit. Owing to the the material we use to prepare the kit, the sample matrix interference may falsely depress the specificity and accuracy of the assay.
Reagent Preparation
All reagents should be brought to room temperature before use.
Standard Reconstitute the 120μl of the standard (2000ng/L) with 120μl of standard diluent to generate a 1000ng/L standard stock solution. Allow the standard to sit for 15 mins with gentle agitation prior to making dilutions. Prepare duplicate standard points by serially diluting the standard stock solution (1000ng/L) 1:2 with standard diluent to produce 500ng/L, 250ng/L, 125ng/L and 62.5ng/L solutions. Standard diluent serves as the zero standard(0 ng/L). Any remaining solution should be frozen at -20°C and used within one month. Dilution of standard solutions suggested are as follows:
| 1000ng/L | Standard No.5 | 120μl Original Standard + 120μl Standard Diluent |
| 500ng/L | Standard No.4 | 120μl Standard No.5 + 120μl Standard Diluent |
| 250ng/L | Standard No.3 | 120μl Standard No.4 + 120μl Standard Diluent |
| 125ng/L | Standard No.2 | 120μl Standard No.3 + 120μl Standard Diluent |
| 62.5ng/L | Standard No.1 | 120μl Standard No.2 + 120μl Standard Diluent |
| Standard Concentration | Standard No.5 | Standard No.4 | Standard No.3 | Standard No.2 | Standard No.1 |
| 2000ng/L | 1000ng/L | 500ng/L | 250ng/L | 125ng/L | 62.5ng/L |
Wash Buffer Dilute 20ml of Wash Buffer Concentrate 30x into deionized or distilled water to yield 500 ml of 1x Wash Buffer. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved.
Summary
1. Prepare all reagents, samples and standards.
2. Add sample and ELISA reagent into each well. Incubate for 1 hour at 37°C.
3. Wash the plate 5 times.
4. Add substrate solution A and B. Incubate for 10 minutes at 37°C.
5. Add stop solution and color develops.
6. Read the OD value within 10 minutes.
Calculation of Result
Construct a standard curve by plotting the average OD for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis and draw a best fit curve through the points on the graph. These calculations can be best performed with computer-based curve-fitting software and the best fit line can be determined by regression analysis.