Double antibody sandwich method 1. Package: With 0.05MPH9. the carbonate pack is buffered to dilute the antibody to a protein content of 1 to 10 ?g/ml. Add 0.1 ml, 4 degrees C overnight, in the reaction holes of each polystyrene plate. The next day, discard the solution in the hole and wash it 3 times with washing buffer for 3 minutes each time.
(for short, washing, the same below). 2. Add sample: Add a certain dilution of the sample to be tested 0.1 ml in the above-mentioned packaged reaction hole, incubate for 1 hour at 37 degrees C. Then wash.
(At the same time do blank hole, negative control hole and positive control hole). 3. Add enzyme-labeled antibodies: In each reaction hole, add a freshly diluted enzyme-labeled antibody (dilution after titration) 0.1 ml.
Incubate 0.5 to 1 hour at 37 degrees C and wash.
4. Add substrate liquid color display: add a temporary preparation of TMB substrate solution 0.1 ml, 37 degrees C 10 to 30 minutes in each reaction hole.
5. End reaction: Add 2M sulphuric acid 0.05 ml to each reaction hole. 6. Results determine: can be on a white background, directly with the naked eye to observe the results: the darker the color inside the reaction hole, the stronger the positive degree, the negative reaction is colorless or very shallow, according to the color is reflected in the depth, to the color of the
Od value scanted: on the ELISA tester, at 450nm (if a BTS color rendering, then 410nm), to blank control hole zero ingested to measure the OD value of each hole, if greater than the specified negative control OD value of 2.1 times, that is, positive.
1. Use the package to be buffered to dilute the known antigen to 1 to 10 mg/ml, per hole plus 0.1 ml, 4 degrees C overnight;
2. Wash 3 times the next day;
3. Add a certain dilution of the sample (unknown antibody) 0.1 ml in the above-mentioned packaged reaction hole, incubate for 1 hour at 37 degrees C, wash;
4. (both blank, negative and positive hole control) in the reaction hole, add a freshly diluted enzyme label second antibody (anti-antibody) 0.1 ml;
5.37 degrees C incubation 35-60 minutes, washing;
6.'The last time you wash with DDW. The remaining steps are the same as the "double antibody sandwich method" of 4, 5, 6.