Rat serum amyloid A (SAA) ELISA kit instructions
Expected application
The ELISA method is used to quantitatively determine the SAA content in rat serum, plasma, cell culture supernatant or other related biological fluids.
Experimental principle
The microplate is coated with the purified antibody to prepare a solid phase carrier, and the sample or standard, the biotinylated anti-SAA antibody, and the HRP-labeled avidin are sequentially added to the microwell coated with the anti-SAA antibody. After washing, the substrate was developed with TMB. TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color by the action of an acid. The shade of the color is positively correlated with the SAA in the sample. The absorbance (OD value) was measured at a wavelength of 450 nm using a microplate reader to calculate the sample concentration.
Kit composition and reagent preparation
1. Enzyme plate: one piece (96 holes)
2. Standard (freeze-dried): 2 bottles, each bottle is diluted to 1ml with the sample dilution before use, and then allowed to stand for more than 10 minutes, then repeatedly reversed/twisted to help dissolve, the concentration is 10 ng /ml, do a series of dilutions (note: do not directly dilute in the plate), diluted to 10 ng / ml, 5 ng / ml, 2.5 ng / ml, 1.25 ng / ml, 0.625 ng / ml , 0.312 ng / ml, 0.156 ng / ml, the sample dilution directly as a standard concentration of 0 ng / ml, prepared within 15 minutes before use. For example, prepare 5 ng/ml standard: take 0.5ml (not less than 0.5ml) 10 ng/ml of the above standard into an Eppendorf tube containing 0.5ml sample dilution, mix well, and so on.
3. Sample dilution: 1 x 20 ml.
4. Test dilution A: 1 x 10 ml.
5. Detect dilution B: 1 x 10 ml.
6. Detection solution A: 1 × 120μl (1:100) diluted with the test solution A 1:100 before use, prepared according to the total amount of each experiment required before dilution (100μl / hole), the actual More 0.1-0.2ml should be prepared during preparation. For example, 10 μl of test solution A plus 990 μl of test diluent A was prepared, gently mixed, and prepared within one hour before use.
7. Detection solution B: 1 × 120 μl / bottle (1: 100) diluted before use to detect dilution B 1:100. The dilution method is the same as the detection solution A.
8. Substrate solution: 1 x 10 ml / bottle.
9. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop solution: 1 x 10 ml / bottle (2N H 2 SO4).
11. Lamination: 5 sheets
12. Instruction manual: 1 copy
Bring your own items
1. Microplate reader (recommended to refer to the instrument instructions for preheating)
2. Micro dosing device and tip, EP tube
3. Distilled or deionized water, new filter paper
Collection and preservation of specimens
1. Serum: Whole blood samples should be placed at room temperature for 2 hours or 4 °C overnight, centrifuged at 1000 xg for 20 minutes, the supernatant can be detected, or the specimens should be stored at -20 ° C or -80 ° C, but should be avoided. Freezing and thawing.
2. Plasma: EDTA or heparin can be used as anticoagulant. Centrifuge at 2 - 8 ° C 1000 xg for 15 minutes within 30 minutes after collection, or store the specimen at -20 °C or -80 °C, but avoid repeated freezing. melt.
3. Cell culture supernatant or other biological specimens: centrifuge at 1000 xg for 20 minutes, take the supernatant for testing, or store the specimen at -20 °C or -80 °C, but avoid repeated freezing and thawing.
4. Sample treatment: serum or plasma samples diluted 100-fold or 100-fold.
Note: The above specimens should be stored at 4 °C for less than 1 week, -20 °C or -80 °C should be sealed and stored, -20 °C should not exceed 1 month, -80 °C should not exceed 2 months; specimen hemolysis will affect the last The test results, so the hemolysis specimen is not suitable for this test.
Steps
Before the start of the experiment, each reagent should be equilibrated to room temperature (the reagent can not be dissolved directly at 37 ° C); when the reagent or sample is diluted, it should be mixed, and 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 so that the diluted sample meets the detection range of the kit, and then multiplied by the corresponding dilution factor.
1. Adding samples: blank holes, standard holes, and sample holes to be tested. Add 100 μl of the sample dilution to the blank well, add 100 μl of the standard or the sample to be tested, and note that there should be no air bubbles. Add the sample to the bottom of the well of the microplate. Try not to touch the wall of the well. Gently shake and mix. The plate was coated or covered with a lid and reacted at 37 ° C for 120 minutes. To ensure the validity of the results, use a new standard solution for each experiment.
2. Discard the liquid, dry it, and do not wash it. 100 μl of the test solution A working solution was added to each well (prepared within one hour before use), and the plate was coated with a membrane and reacted at 37 ° C for 60 minutes.
3. After 60 minutes of incubation, discard the liquid in the well, dry it, wash the plate 3 times, soak for 1-2 minutes each time, about 400μl/well, dry (can also pat the liquid in the well) .
4. Add 100 μl of test solution B working solution (with the same test A working solution) to each well, and react with the plate at 37 ° C for 60 minutes.
5. After 60 minutes of incubation, discard the liquid in the well, dry and wash the plate 5 times, soak for 1-2 minutes each time, 350 μl/well, and dry (can also pat the liquid in the well).
6. Add 90μl of substrate solution to each well in sequence, and add the membrane to the plate at 37°C to avoid light. (In 30 minutes, the first 3-4 holes of the standard can be seen by the naked eye.) The 3-4 hole gradient is not obvious and can be terminated).
7. Add 50 μl of the stop solution to each well in sequence to terminate the reaction, at which time the blue color turns yellow. The order of addition of the stop liquid should be as close as possible to the order in which the substrate liquid is added. In order to ensure the accuracy of the experimental results, the substrate should be added as soon as possible after the reaction time.
8. The optical density (OD value) of each well was measured sequentially at 450 nm using an enzyme-linked instrument. The test was performed immediately after the addition of the stop solution.
Note:
1. Reagent preparation: All reagents must reach room temperature before use. Please keep the reagents as required according to the instructions. Use disposable tips during the experiment to avoid cross-contamination.
2. Loading: When adding or adding reagents, please note that when taking specimen/standard, enzyme conjugate or substrate, if the time interval between the first hole and the last hole is too large, it will be This results in different "pre-incubation" times, which significantly affects the accuracy and repeatability of the measurements. The loading time (including the standard and all samples) is preferably controlled within 10 minutes. If the number of specimens is large, it is recommended to use a multi-channel pipette.
3. Incubation: In order to prevent the sample from evaporating, put the reaction plate in a closed box covered with a damp cloth, and cover or cover the plate with the enzyme plate to avoid evaporation of the liquid; the next step should be carried out as soon as possible after washing the plate, any At all times, the plate should be kept dry; the given incubation time and temperature should be strictly observed.
4. Washing: The washing liquid remaining in the reaction well during the washing process should be fully patted on the filter paper. Do not put the filter paper directly into the reaction hole to absorb water. At the same time, remove the residual liquid and fingerprints at the bottom of the plate to avoid affecting the last enzyme. Standard reading.
5. Reagent preparation: Detection A and Detection B should be centrifuged a few times or less before use to deposit the liquid on the tube wall or cap to the bottom of the tube. Standard product, test solution A working solution, test solution B working solution should be configured according to the required amount and prepared with the corresponding dilution solution, which should not be confused. Please accurately configure the standard and working fluid, try not to make a small amount of configuration (such as not to be less than 10μl when taking the test solution A) to avoid the concentration error caused by inaccurate dilution; do not reuse the diluted standard, Detect solution A working solution or test solution B working solution.
6. Control of reaction time: After adding the substrate, please observe the color change of the reaction well regularly (for example, every 10 minutes). If the color is darker, please add the stop solution in advance to terminate the reaction to avoid the reaction being too strong and affect the microplate reader. Optical density reading.
7. Substrate: Keep the substrate away from light and avoid direct exposure to strong light during storage and incubation.
It is recommended to set a double-hole measurement when testing samples to ensure the accuracy of the test results.
If the content of the substance to be tested in the specimen is too high, please dilute it before measuring it. When calculating, multiply by the dilution factor.
Washing method
1. Manual washing method: suck (not touch the wall) or pry off the liquid in the microplate; place several layers of absorbent paper on the test bench, and take the microplate with the microplate down several times; the recommended washing buffer At least 0.3 ml of the injection hole, soak for 1-2 minutes, repeat this process several times as needed.
2. Automatic washing: If there is an automatic washing machine, it should be used in the formal experiment after skilled use.
Specificity
The kit detects both recombinant or native rat SAA and does not cross-react with other related proteins.
Calculation
The concentration of the standard is plotted on the ordinate (logarithmic coordinates) and the OD is plotted on the abscissa (logarithmic coordinates). A standard curve is drawn on the logarithmic scale paper. It is recommended to use professional production curve 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 multiply by the dilution factor; or calculate the regression equation of the standard curve by using the concentration of the standard and the OD value. Substituting the OD value of the sample into the equation, calculating the sample concentration, and multiplying by the dilution factor, is the actual concentration of the sample.
Detection range: 0.156 ng/ml - 10 ng/ml
Minimum detection limit: 0.039 ng/ml
Description
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, and the reagents should be protected from microbial contamination, as proteolytic enzyme interference can lead to erroneous results.
2. Kit storage: Short-term (within 1 week) is based on the label on the label. For long-term storage, store all reagents at -20 °C.
3. The concentrated washing liquid will be salted out. When it is diluted, it can be heated and dissolved in the water bath.
4. There may be a little water-like substance in the opening of the enzyme-linked plate. This is normal and will not affect the experimental results.
5. All samples should be managed and the samples and test equipment processed in accordance with the prescribed procedures.
6. Validity: 6 months.
7. These instructions apply to the 48T kit, but all reagents in the 48T kit are halved.
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