Human Vascular Endothelial Growth Factor (VEGF165) ELISA Kit Instructions
This kit is only for in vitro research!
Intended application
Quantitative determination of VEGF165 in human serum, plasma, cell culture supernatant or other related biological fluids by ELISA.
Experimental principle
Coat the microplate with purified VEGF165 antibody to make a solid-phase carrier. Add specimens or standards, biotinylated VEGF165 antibody, HRP-labeled avidin to the microwells in sequence, and wash the substrate with TMB) color development. 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 VEGF165 in the sample. Measure the absorbance (OD value) at 450nm with a microplate reader to calculate the sample concentration.
Kit composition and reagent preparation
1. Microplate: one piece (96 wells)
2. Standard product (lyophilized product): 2 bottles, please prepare within 15 minutes before use. Each bottle is diluted to 1ml with sample diluent, capped
Let stand at room temperature for about 10 minutes, while inverting / shaking repeatedly to help dissolve. Its concentration is 5,000 pg / ml, and then do serial dilutions (Note: Do not directly perform multiple dilutions in the plate). 5,000 pg / ml, 2,500 pg / ml, 1,250 pg / ml, 625 pg / ml, 312 pg / ml, 156 pg / ml, 78 pg / ml, the sample diluent is directly used as blank well 0 pg / ml. To prepare 2,500 pg / ml standard: Take 0.5ml (not less than 0.5ml) of the above standard at 5,000 pg / ml and add it to the Eppendorf tube containing 0.5ml of sample diluent, mix well. The rest of the concentration can be deduced by analogy.
3. Sample diluent: 1 × 20ml.
4. Test dilution A: 1 × 10ml.
5. Test diluent B: 1 × 10ml.
6. Detection solution A: 1 × 120 per bottle (1: 100). Before use, dilute with test diluent A 1: 100 (eg: 10 test
Solution A / 990 Detection Diluent A), mix well, according to the pre-calculated total amount required for each experiment before dilution
Preparation (100 / well), more 0.1-0.2ml should be prepared during actual preparation.
7. Detection solution B: 1 × 120 per bottle (1: 100). Dilute 1: 100 with test dilution B immediately before use. Dilution method same inspection
Test solution A.
8. Substrate solution: 1 × 10ml / bottle.
9. Concentrated washing liquid: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop solution: 1 × 10ml / bottle (2 mol / L H2SO4).
11. Lamination: 5 sheets
12. Instruction manual: 1 copy
Bring your own items
1. Microplate reader (It is recommended to preheat the instrument before use)
2. Micro dosing device and pipette tip, EP tube
3. Distilled or deionized water, filter paper
Collection and preservation of specimens
1. Serum: Whole blood samples should be left at room temperature for 2 hours or 4 overnight, centrifuged at 1000 g for 20 minutes, and the supernatant can be taken
Test or store the supernatant at -20 or -80, but avoid repeated freezing and thawing.
2. Plasma: EDTA or heparin can be used as anticoagulant. Centrifuge at 1000 g for 15 minutes within 30 minutes after specimen collection.
Take the supernatant for testing, or store the supernatant at -20 or -80, but avoid repeated freezing and thawing.
3. Cell culture supernatant or other biological specimens: centrifuge at 1000 g for 20 minutes, take the supernatant for detection, or store the supernatant at -20 or -80, but avoid repeated freezing and thawing.
Note: The above specimens should be sealed and stored, 4 should be stored for less than 1 week, -20 should not exceed 1 month, -80 should not exceed 2 months; specimen hemolysis will affect the final test results, so hemolysis specimens should not be tested .
Steps
Before starting the experiment, all reagents should be equilibrated to room temperature (reagents cannot be dissolved directly in 37); when preparing reagents or samples,
All need to be mixed thoroughly, try to avoid foaming when mixing. The sample content should be predicted before the experiment. If the sample concentration is too high, the sample should be diluted to make the diluted sample meet the detection range of the kit, and then multiplied by the corresponding dilution factor when calculating.
1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. Add 100% of sample diluent to blank wells
The standard or the sample to be tested is 100. Be careful not to have air bubbles. When adding the sample, add the sample to the bottom of the microtiter plate and try not to touch it.
The walls of the wells were gently shaken and mixed. The microtiter plate was covered or covered, and incubated at 37 for 2 hours. To ensure that the experimental results are valid
For each experiment, please use a new standard solution.
2. Discard the liquid and dry it without washing. Add testing solution A working solution 100 to each well (prepared before use)
Cover with film and incubate at 37 for 1 hour.
3. Discard the liquid in the hole, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, about 400 per hole, spin dry (also light
Pat to dry the liquid in the hole).
4. Add testing solution B working solution (prepared before use) 100 to each well, cover with film, and incubate at 37 for 1 hour.
5. Discard the liquid in the hole, spin dry, wash the plate 5 times, the method is the same as step 3.
6. Add substrate solution 90 for each well, enzyme label plate and film 37 to avoid color development (reaction time is controlled at 15-30 minutes, when
The first 3-4 wells of the standard wells have a clear gradient blue, and the latter 3-4 wells can be terminated when the gradient is not obvious.
7. Add 50% stop solution to each well to stop the reaction. At this time, the blue color turns to yellow. The order of adding the stop solution should be as close as possible to the substrate
The order of adding the liquid is the same.
8. Immediately measure the optical density (OD value) of each well with a microplate reader at 450nm wavelength.
Note:
1. Preparation of reagents: All reagents should be equilibrated to room temperature before use. Please save the reagents according to the instructions immediately after use.
Please use disposable tips during the experiment to avoid cross contamination.
2. Add sample: when adding sample or adding reagent, if the time interval between the first well and the last well is too large, it will cause
The same "pre-incubation" time, which obviously affects the accuracy and repeatability of the measured values. Sample loading time (including standard
Products and all samples) is best controlled within 10 minutes. It is recommended to set up multiple holes for experiments.
3. Incubation: In order to prevent the sample from evaporating, place the enzyme label plate with a cover or membrane in the wet box during the experiment to avoid liquid evaporation;
After washing the plate, the next step should be carried out as soon as possible. At any time, the microplate should be avoided in a dry state;
Fixed incubation time and temperature.
4. Washing: the washing liquid remaining in the reaction well during the washing process should be patted dry on the filter paper, do not put the filter paper directly into the reaction well
Absorb water, and at the same time eliminate the residual liquid and fingerprints on the bottom of the plate to avoid affecting the final reading of the microplate reader.
5. Preparation of reagents: Before using A, please shake the hand a few times or centrifuge for a while to prevent the wall of the tube
Or the liquid from the bottle cap is deposited to the bottom of the tube. Standard products, testing solution A working fluid, testing solution B working fluid
The amount of preparation and use, and use the corresponding dilution to prepare, not to be confused. Please accurately prepare standard products and working fluid
Do not prepare in small amounts (for example, when drawing test solution A, it should not be less than 10 at a time) to avoid inaccurate dilution
The resulting concentration error; do not reuse the diluted standard, test solution A working solution or test solution B
Working fluid.
6. Control of reaction time: after adding the substrate, please observe the color change of the reaction well regularly (for example, every 10 minutes), such as
The color is darker, please stop the reaction by adding stop solution in advance to avoid the reaction is too strong and affect the optical density reading of the microplate reader.
7. Substrate: Please keep the substrate away from light, and avoid direct exposure to strong light during storage and incubation.
Washing method
1. Manual plate washing method: Inject at least 0.4ml of the recommended washing buffer into the well, soak for 1-2 minutes, and suck off (not possible
Touch the wall of the plate) or shake off the liquid in the microplate, place a few layers of absorbent paper on the experimental Table, and tap the microplate down with a few strokes
Times; repeat this process as many times as needed.
2. Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used proficiently.
Specificity
The kit can detect recombinant or natural human VEGF165 at the same time, and does not cross-react with other related proteins.
Calculation
Draw the OD value of each standard product after deducting the OD value of the blank hole (seven-point diagram). If multiple holes are set, the average value should be used for calculation. Taking the concentration of the standard product as the ordinate (logarithmic coordinate) and the OD value as the abscissa (logarithmic coordinate), draw a standard curve on the logarithmic coordinate paper. It is recommended to use professional curve making software for analysis, such as curve expert 1.3, find the corresponding concentration from the standard curve according to the OD value of the sample, and then multiply it by the dilution factor; or use the concentration and OD value of the standard to calculate the regression equation of the standard curve , Substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor, which is the actual concentration of the sample.
Detection range: 78 pg / ml-5,000 pg / ml, please draw the following concentration values ​​for drawing standard curve: 5,000 pg / ml, 2,500 pg / ml, 1,250 pg / ml, 625 pg / ml, 312 pg / ml, 156 pg / ml, 78 pg / ml.
Minimum detection limit: 20 pg / ml
Explanation
1. Avoid exposing the reagent to strong light during storage and incubation. All reagent bottle caps must be tightly closed to prevent evaporation and microbial
Contamination, because the interference of proteolytic enzymes will lead to wrong results.
2. Storage of the kit: Please save the standard, detection solution A and detection solution B at -20 as soon as possible after receiving the kit
For short-term storage, please set to 4; for long-term storage, set to -20. After opening, the microplate should be sealed and added with desiccant and stored in
-20 to avoid moisture.
3. Salt will precipitate out in the concentrated washing liquid, which can be heated and dissolved in the water bath when diluted.
4. There may be a little water-like substance 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.
5. Validity: 6 months.
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Human Vascular Endothelial Growth Factor (VEGF165) ELISA Kit Instructions
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