Common methods for ELISA kits - sandwich method and indirect method - Database & Sql Blog Articles

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Kaixin micro test

Test probe P100-M3

Shanghai Win-win bio-elisa kit is commonly used in two methods. I hope I can help you.
(a) double antibody sandwich method

1. Coat: The antibody was diluted to a protein content of 1 to 10 μg/ml with 0.05 MPH 9. 牰 carbonate coating buffer. 0.1 ml was added to the reaction well of each polystyrene plate at 4 ° C overnight. 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. Loading: Add 0.1 ml of the sample to be tested diluted in the above-mentioned coated reaction wells, and incubate at 37 ° C for 1 hour. Then wash. (Do blank holes, negative control wells and positive control wells at the same time).

3. Add enzyme-labeled antibody: Add 0.1 ml of freshly diluted enzyme-labeled antibody (diluted titration) to each well. Incubate at 37 ° C for 0.5 to 1 hour and wash.

4. Add substrate liquid to develop color: Add 0.1 ml of temporarily prepared TMB substrate solution to each reaction well at 37 ° C for 10 to 30 minutes.

5. Stop the reaction: Add 0.05 ml of 2M sulfuric acid to each reaction well.

6. Judgment of results: The results can be observed directly on the white background with the naked eye: the darker the color in the reaction well, the stronger the positive degree, the negative reaction is colorless or very light, according to the depth of the color, with "+", The "-" sign indicates. The OD value can also be measured: on the ELISA detector, at 450 nm (if the color is developed by ABTS, 410 nm), the OD value of each well is measured after zero adjustment of the blank control well, if it is greater than 2.1 times the OD value of the specified negative control. Is positive.

(2) Indirect law

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

2. Wash 3 times the next day;

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

4. (while doing blank, negative and positive well control) in the reaction well, add freshly diluted enzyme-labeled secondary antibody (anti-antibody) 0.1ml;

Incubate at 5.37 ° C for 35-60 minutes, wash;

6. 'The last time wash with DDW.

The remaining steps are the same as the "double antibody sandwich method" 4, 5, and 6.