ELISA steps

Method 1: Double antibody sandwich method for detecting unknown antigens:

1. Coating: Dilute the antibody with 0.05M PH9 and carbonate coating buffer to a protein content of 1 ～ 10μg / ml, add 0.1ml to the reaction well of each polystyrene plate, and 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.

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. OD value can also be measured: on ELISA detector, at 450nm (410nm if ABTS color is developed), the OD value of each well is measured after zero adjustment with a blank control well, if it is greater than 2.1 times the OD value of the specified negative control , That is positive.

Method 2: Indirect method for detecting unknown antibodies:

Dilute the known antigen with coating buffer to 1-10μg / ml, add 0.1ml per well, and overnight at 4 ℃. Wash 3 times the next day. Add a certain dilution of the test sample (unknown antibody) 0.1ml to the coated wells, incubate at 37 ° C for 1 hour, and wash. (Make blank, negative and positive well control at the same time) Add 0.1ml of fresh diluted enzyme-labeled secondary antibody (anti-antibody) to the reaction well, incubate at 37 ℃ for 30 to 60 minutes, wash, and wash with DDW one last time. The remaining steps are the same as 4, 5 and 6 of the "double antibody sandwich method".

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