This kit is for research use only
ELISA method was used to quantitatively determine the secretion of vascular endothelial growth factor (EG-VEGF) in human serum, plasma or other related fluids.
Experimental principle The kit uses double antibody sandwich enzyme immunoassay to determine the level of EG-VEGF in the specimen. The microtiter plate was coated with purified antibody to make a solid-phase antibody. EG-VEGF antigen, biotinylated anti-human EG-VEGF antibody, and HRP-labeled avidin were added to the monoclonal antibody-coated microwells in turn After thorough washing, color was developed with substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with EG-VEGF in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated.
1. ELISA plate: one piece (96 wells)
Standard product (lyophilized product): 2 bottles, each bottle is diluted with sample diluent to 1ml before use, and its concentration is 1000 pg / mL, after serial dilution, it is diluted to 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31 pg / mL, 15.6 pg / mL, the original solution is directly used as the highest standard concentration, the sample dilution is directly used as the standard concentration 0 pg / mL, before use Prepare within 15 minutes.
For example, to prepare a 500 pg / mL standard: Take 0.5ml 1000 pg / mL of the above standard into an Eppendorf tube containing 0.5ml of sample diluent, mix well, and the rest of the concentration can be deduced by analogy.
2. Sample diluent: 1 Ã— 20ml / bottle.
3. Test the diluent A: 1 Ã— 10ml / bottle.
4. Test dilution B: 1 Ã— 10ml / bottle.
5. Detection solution A: 1 Ã— 120ul / bottle (1: 100) diluted with detection diluent A 1: 100 before use, prepared according to the pre-calculated total amount required for each experiment before dilution (100ul per well) , The actual preparation should be more 0.1-0.2ml. For example, 1ul detection solution A plus 99ul detection dilution A is prepared in proportion, mix gently and prepare within one hour before use.
6. Test solution B: 1 Ã— 120ul / bottle (1: 100) is diluted with test diluent B1: 100 before use. The dilution method is the same as that of Test Solution A.
7. Substrate solution: 1 Ã— 10ml / bottle.
8. Concentrated washing solution: 1 Ã— 30ml / bottle, each bottle is diluted 25 times with distilled water.
9. Stop solution: 1 Ã— 10ml / bottle (2N H2SO4).
Collection and preservation of specimens
1. Serum: Please leave the specimen at room temperature for 2 hours or overnight at 4 Â° C and centrifuge at 1000 xg for 20 minutes. Take the supernatant for testing, or store the specimen at -20 Â° C, but avoid repeated freezing and thawing.
2. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge at 1000 xg for 15 minutes at 2-8 Â° C within 30 minutes after the specimen is collected, or store the specimen at -20 Â° C, but repeated freezing and thawing should be avoided.
Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested.
Operation steps Each reagent is equilibrated to room temperature before use.
1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. In addition to the blank wells, add 100 ul of standard solution or sample to be tested to the remaining wells, taking care not to have air bubbles, mix gently, cover the enzyme plate, and react at 37 Â° C for 120 minutes.
2. Discard the liquid and spin dry without washing.
3. Add 100ul of detection solution A working solution to each well at 37 â„ƒ for 60 minutes. Wash the plate 3 times, 350ul / per well, spin dry.
4. Add 100ul of detection solution B working solution to each well at 37 Â° C for 60 minutes, wash the plate 5 times and spin dry.
5. Add 90ul of substrate solution to each well in sequence, and develop the color for 30 minutes at 37 Â° C in the dark (at this time, the first 3-4 wells of the standard product have a clear blue gradient, and the latter 3-4 wells are not obvious).
6. Add 50ul of stop solution to each well in sequence to stop the reaction (at this time, the blue color turns to yellow). The optical density (OD value) of each well was measured sequentially with an enzyme-linked instrument at a wavelength of 450 nm.
1. One hole is left for each experiment as a blank zero-adjusting hole. No reagents are added to this hole, only the substrate solution and 2NH2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring.
2. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test.
Plate washing method Manual plate washing method: suck (not touch the wall) or shake off the liquid in the microplate; put a few layers of absorbent paper on the experimental table, and tap the microplate down several times with force; the recommended wash buffer Inject at least 0.4ml of solution into the hole and soak for 1-2 minutes. Repeat this process several times as needed.
Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.
Specificity This kit can detect recombinant or natural human EG-VEGF at the same time, and does not cross-react with other related proteins.
Calculate the standard concentration as the abscissa (logarithmic coordinate), the OD value is the ordinate (ordinary coordinate), draw a standard curve on the semi-logarithmic coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample ; Multiply by the dilution factor; or use the standard concentration and OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual concentration of the sample .
1. The washing process is very important, inadequate washing is easy to cause false positives.
2. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
3. Please make a standard curve at the same time of each measurement, it is best to make a double hole.
4. If the content of the substance to be tested in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor.
5. When preparing the standard and the working solution of the test solution, please prepare it with the corresponding diluent, not to be confused.
6. Please keep the substrate away from light.
1. Avoid exposing the reagent to strong light during storage and incubation. All reagent bottle caps must be tightly closed to prevent evaporation and contamination. Reagents should be protected from microbial contamination, because the interference of proteolytic enzymes will lead to erroneous results.
2. Aspirate the reagents carefully and strictly observe the given incubation time and temperature. Please note that when drawing samples / standards, enzyme conjugates or substrates, if the time interval between the first well and the last well is too large, it will result in different "pre-incubation" time, which obviously Affect the accuracy and repeatability of the measured value. Moreover, insufficient washing will affect the test results.
3. Storage of the kit: some reagents are stored at -20 Â° C, and some reagents are stored at 2-8 Â° C, depending on the label.
4. Salt will precipitate out in the concentrated washing liquid, and it can be heated and dissolved in the water bath when diluted.
5. There may be some water-like substances in the well of the enzyme-linked plate just opened. This is a normal phenomenon and will not have any impact on the experimental results.
6. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.
7. All samples should be managed, and the samples and testing devices should be processed according to the prescribed procedures.
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