How To Operate The Elisa Kit

Double antibody sandwich method

1. Coating: Dilute the antibody to a protein content of 1-10 μg/ml with 0.05MPH 9. Oxycarbonate Coating Buffer. Add 0.1 ml to the reaction well of each polystyrene plate, overnight at 4°C. The next day, the solution in the wells was discarded and washed three times with wash buffer for 3 minutes each. (referred to as washing, the same below).

2. Add sample: Add 0.1ml of a certain diluted sample to be tested to the above coated reaction well, and incubate at 37°C for 1 hour. Then wash. (At the same time, make blank wells, negative control wells and positive control wells).

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 solution for color development: add 0.1 ml of temporarily prepared TMB substrate solution to each reaction well, and incubate at 37°C for 10 to 30 minutes.

5. Termination of 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 shallow. "-" sign indicates. OD value can also be measured: on the ELISA detector, at 450nm (410nm if ABTS is used for color development), measure the OD value of each well after zeroing with the blank control well, if it is greater than 2.1 times the OD value of the specified negative control , which is positive.

Indirect method

1. Dilute the known antigen to 1-10 μg/ml with coating buffer, add 0.1 ml to each well, and keep overnight at 4°C;

2. Wash 3 times the next day;

3. Add 0.1 ml of a certain diluted sample to be tested (unknown antibody) to the above-mentioned reaction wells, incubate at 37°C for 1 hour, and wash;

4. (Do blank, negative and positive well controls at the same time) Add 0.1ml of freshly diluted enzyme-labeled secondary antibody (anti-antibody) to the reaction well;

5. Incubate at 37°C for 35-60 minutes, wash;

6.' Final wash with DDW.

The remaining steps are the same as 4, 5, and 6 of the "Double Antibody Sandwich Method".