In the sandwich ELISA method, the checkerboard titration method can be used to select the working concentration of the coating antibody and the enzyme-labeled antibody simultaneously. Coated on the ELISABAN plate, each concentration was coated with three longitudinal rows and washed.
2 Add a strong positive antigen solution to each coating well in one horizontal row, a weak positive antigen solution in the other horizontal row, and a negative control solution in the third horizontal row, incubate and wash the strips.
3 Dilute the enzyme-labeled antibody to three concentrations with diluent
Add to each column of each coating concentration separately, incubate and wash.
4 Add substrate to develop color, after adding acid to stop the reaction, read A value.
5 As the optimal condition, the A value of the strong positive antigen solution is about 0.8, and the A value of the negative reference solution is less than 0.1. According to this, the working concentration of the coated antibody and the enzyme-labeled antibody is selected. The pH and ionic strength of the coating buffer also have an effect on adsorption. The pH of the protein-coated buffer solution should be 5 alkaline, which can achieve a good adsorption effect at pH = 7 ~ 10, such as the commonly used buffer solution is carbonate buffer solution (PH = 9.6)
The buffer also affects the sensitivity of certain methods. When coated with lipids and viral antigens, it is necessary to add sodium deoxycholate, which partially denatures the coated antibody F segment to increase sensitivity.
Some monoclonal antibodies are difficult to adsorb to the solid phase, and different buffers and buffer concentrations and pHs need to be tried. In addition, attention should be paid to the coating conditions and the concentration of the coating antigen and antibody.
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