Shanghai ancient flower biology tells you: plant carbonic anhydrase (CA) ELISA kit instructions
First, the operation steps
Dilution: This kit provides one original standard, which is diluted several times in sequence.
Adding samples: blank holes are respectively set (the blank control wells are not added with the sample and the enzyme standard reagent, and the other steps are the same), the standard holes, and the sample holes to be tested. Accurately load 50 μl of the standard on the enzyme-labeled plate, add 40 μl of the sample dilution to the well to be tested, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix.
Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.
Dosing: 30 times (20 times of 48T) concentrated washing solution diluted with distilled water 30 times and used
Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.
Add enzyme: 50 μl of enzyme labeling reagent was added to each well, except for blank wells.
Incubation: The operation is the same as 3.
Washing: The operation is the same as 5.
Color development: Add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake and mix, and develop color at 37 ° C for 10 minutes.
Termination: 50 μl of stop solution was added to each well to terminate the reaction (in this case, the blue color turned yellow).
Measurement: The absorbance (OD value) of each well was measured in sequence with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution.
Second, matters needing attention
1. The kit should be taken out from the refrigerated environment and allowed to equilibrate for 15-30 minutes at room temperature. If the enzyme label is unsealed after opening, the slats should be stored in a sealed bag.
2. Concentrated washing liquid may crystallize. When diluted, it can be heated and dissolved in the water bath. The washing will not affect the result. 3. The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun.
4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the first hole of the standard well), please first dilute the sample dilution with a certain multiple (n times) and then measure it. When calculating, multiply the total dilution by the total dilution. Multiple (×n×5).
5. The sealing film is for one-time use only to avoid cross-contamination.
6. Keep the substrate away from light.
7. Strictly follow the instructions in the manual. The test results must be based on the reading of the microplate reader.
8. All samples, washings and various wastes should be treated as infectious materials.
9. The different batch components of this reagent must not be mixed.
10. In the case of an English manual, the English manual shall prevail.
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