1. Dilution and loading of standard products: 10 wells of standard wells are placed on the enzyme labeling board, 100 μl of standard is added to the first and second holes, and then standard is added to the first and second holes. 50 μl of the dilution solution, and mix; then, 100 μl of each of the first hole and the second hole are respectively added to the third hole and the fourth hole, and then 50 μl of the standard dilution solution is added to the third and fourth holes, respectively, and mixed; Then, 50 μl of each of the third hole and the fourth hole are discarded, and 50 μl of each is added to the fifth and sixth holes, respectively, and 50 ul of the standard dilution solution is added to the fifth and sixth holes, respectively, and mixed; After mixing, 50 μl from each of the fifth and sixth wells was added to the seventh and eighth wells respectively, and then 50 μl of the standard dilution was added to the seventh and eighth wells, and the mixture was mixed from the seventh and the seventh. 50 μl of each of the eight wells was added to the ninth and tenth holes, and 50 μl of the standard dilution was added to the ninth and tenth holes, respectively, and 50 μl of each of the ninth and tenth holes was discarded. (The amount of each well was 50 μl after dilution, and the concentrations were 60 ng/ml, 40 ng/ml, 20 ng/ml, 10 ng/ml, 5 ng/ml, respectively).
2. Loading: Set blank holes separately (the blank control wells are not added with the sample and the enzyme standard reagent, the other steps are the same), and the sample holes to be tested. Add 40 μl of the sample dilution to the sample well to be tested on the plate, 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.
3. Incubation: After sealing with a sealing film, incubate at 37 ° C for 30 minutes.
4. Dosing: Dilute 30 (20 times of 48T) concentrated washing solution with distilled water 30 (20 times of 48T) and set aside.
5. 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.
6. Add enzyme: 50 μl of enzyme labeling reagent was added to each well, except for blank wells.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. 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 15 minutes.
10. Termination: 50 μl of stop solution was added to each well to terminate the reaction (at this time, the blue color turned yellow).
11. Measurement: The absorbance (OD value) of each well was measured sequentially 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.
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