1. Coating: Dilute the antibody with a 0.05M PH9. Carbonate coating buffer to a protein content of 1 to 10 μg / ml. Add 0.1ml to the reaction well of each polystyrene plate, overnight at 4 ℃. The next day, the solution in the well was discarded and washed 3 times with washing buffer for 3 minutes each time. (Referred to as washing, the same below).
2. Sample addition: add 0.1ml of the diluted test sample to the above-mentioned coated wells and incubate at 37 ℃ for 1 hour. Then wash. (Make blank wells, negative control wells and positive control wells at the same time).
3. Add enzyme-labeled antibody: add 0.1ml of freshly diluted enzyme-labeled antibody (dilution after titration) to each reaction well. Incubate at 37 ° C for 0.5 to 1 hour and wash.
4. Add substrate liquid to develop color: add 0.1ml of temporarily prepared TMB substrate solution to each reaction well at 37 ℃ for 10-30 minutes.
5. Terminate the reaction: Add 0.05ml of 2M sulfuric acid to each reaction well.
6. Judgment of results: The results can be directly observed with the naked eye on a white background: the darker the color in the reaction well, the stronger the positive degree, and the negative reaction is colorless or extremely light. "-" Indicates. O · D value can also be measured: On the ELISA detector, at 450nm (if ABTS color development, then 410nm), the blank control well is zeroed and the O · D value of each well is measured, if it is greater than the specified negative control OD 2.1 times the value is positive.
1. During the formal test, the test conditions should be controlled by the positive control and the negative control, respectively, and the samples to be tested should be made in duplicate to ensure the accuracy of the experimental results. Sometimes the background is high, indicating a non-specific reaction, which can be blocked with sheep serum, rabbit serum or BSA.
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