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This ELISA kit can be applied to the in vitro quantitative determination of Mouse VEGF-A concentrations in serum, plasma and other biological fluids.
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Mouse VEGF-A. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse VEGF-A and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Mouse VEGF-A, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Mouse VEGF-A. The concentration of Mouse VEGF-A in samples can be calculated by comparing the OD of the samples to the standard curve.
|Detection Range||31.25—2000 pg/mL|
|Sample Volume Required Per Well|
This kit recognizes some recombinant and natural Mouse VEGF-A. No significant cross-reactivity or interference was observed.
For convenience in result calculation, absorbance can be used as ordinate and standard concentrations as abscissa. The standard curve provided in the manual is only for reference, and experimenters should draw the standard curve based on data of themselves.
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high leve Mouse VEGF-A were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Mouse VEGF-A were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||5.05||4.95||5.40||5.08||4.97||5.31|
The recovery of Mouse VEGF-A spiked to three different levels in samples throughout the range of the assay in various matrices was evaluated.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||98-108||101|
|Cell culture media (n=5)||99-109||104|
Samples were spiked with high concentrations of Mouse VEGF-A and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
|Serum (n=5)||EDTA plasma (n=5)||Cell culture media (n=5)|
The unopened kit can be stored at 4℃ for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions since the kit is received.
|Micro ELISA Plate(Dismountable)||8 wells ×12 strips||-20℃, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100×)||1 vial, 120 μL|
|Concentrated HRP Conjugate (100×)||1 vial, 120 μL||-20℃(shading light), 6 months|
|Reference Standard & Sample Diluent||1 vial, 20 mL||4℃, 6 months|
|Biotinylated Detection Ab Diluent||1 vial, 14 mL|
|HRP Conjugate Diluent||1 vial, 14 mL|
|Concentrated Wash Buffer (25×)||1 vial, 30 mL|
|Substrate Reagent||1 vial, 10 mL||4℃(shading light)|
|Stop Solution||1 vial, 10 mL||4℃|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
Note: All reagent bottle caps must be tightened to prevent evaporation and microbial pollution.
The volume of reagents in partial shipments is a little more than the volume marked on the label, please use in measuring instead of directly pouring.
1.Add 100 μL of standard or sample to each well.
2.Remove the liquid.
3.Add 100 μL Biotinylated Detection Ab. Incubate 1 hour at 37℃.Aspirate and wash 3 times.
4.Add 100 μL HRP Conjugate. Incubate 30 min at 37℃.Aspirate and wash 5 times.
5.Add 90 μL of Substrate Reagent. Incubate for 15 min at 37℃.
6.Add 50 μL of Stop Solution.
7.Read the OD at 450 nm immediately. Calculation of the results.