The binding between an antigen and an antibody can be affected by factors such as temperature, pH, the components of the buffers, and the relative concentration of antigen / antibody.
The antigen concentration is a factor that can hardly be modified, therefore it is necessary to titrate the antibodies by means of a series of dilutions, to determine the optimal working concentration in each case. This must be done for each application, and taking into account the experimental conditions of each test.
Why Titrate The Antibodies?
The main objectives pursued with the antibody titration are:
- Optimize the consumption of the product, adjusting the amount of reagent and therefore reducing costs.
- Reduce noise due to non-specific binding of the antibody, as well as low affinity binding, due to excess antibody in the medium.
- Increase the sensitivity of the assay, as too low antibody concentrations could compromise the signal.
How To Titrate Antibodies
Antibody titration is a necessary step to optimize dilution of these bioreactants for each assay.
The optimal concentration will be that which provides a better signal / noise ratio. This concentration must be determined individually for each test, and for this the most frequently used method is that of serial dilutions.
To carry out an antibody titration experiment , it is necessary to set the incubation time as well as the experimental dilutions of the antibody to be tested. Each of these dilutions should be tested on the same type of sample to maintain experimental conditions.
Usually, the consistency between batches of commercially available antibodies is usually high, so once titrated, it is not usually necessary to repeat this process in the future. However, and particularly in the case of polyclonal antibodies , it is recommended to titrate again in case of detecting unexpected changes in the results.
Recommended Dilutions To Titrate Antibodies
Even in those cases where the manufacturer indicates the recommended working dilution in the technical sheet, it is recommended to titrate the antibodies in our laboratory. In these cases, we will start from the dilution recommended by the manufacturer, and we will carry out the test with that and at least four other dilutions: 2 major and 2 minor.
In case we do not have a recommended dilution, we can be guided from the following table, where the recommended dilutions are indicated depending on the technique and the source of antibodies.