Regardless of whether the antibodies are purified or not (sera, culture supernatants, ascites fluid, etc.), it is usual to have to dilute them before using them in the corresponding application in order to optimize their final concentrations.
In this entry we discuss the importance of determining the optimal working concentration for a given antibody, and explain the differences between the concepts of dilutions and antibody titre.
The reason why it is necessary to dilute our antibody sample to the optimum concentration for our assay is simple: if we use it at too high concentrations, interference and background noise will be greater. In addition, we will spend more reagent than necessary, increasing the cost of the experiment.
Conversely, if we use too low antibody concentrations, the antigen of interest may not be detected, leading to false negative results.
For all of the above, it is essential to optimize the dilution of our antibody for each of the applications in which it will be used, in order to obtain the best signal-to-noise ratio.
OPTIMIZE ANTIBODY DILUTION
The optimal working concentration for each particular antibody must be determined based on each specific application or immunoassay, and for certain experimental conditions (temperature, pH, buffer, etc.).
But how can we establish the optimal concentration for a given antibody in a specific technique? Very easy, by means of a titration experiment using serial dilutions of the antibody stock solution. Each of these dilutions will be tested against the test sample, measuring the results with the corresponding detection method until reaching the dilution at which a positive result is no longer obtained.
With this titration experiment we will obtain the so-called antibody titer , which will correspond to the highest dilution in the series to which a positive result has been obtained.
The antibody titer is a concept related to dilution of antibodies, but technically different. The antibody titer is the highest dilution of the antibody at which the immunoassay in question still yields a positive result.