Depending on the antigenic target being studied and the application being used, it may be advisable to employ complementary strategies during antibody validation. These approaches can provide vital information regarding antibody specificity or functionality and can be carefully tailored to the biological nature of the target as well as to the exacting requirements of the downstream assay.
For antigenic targets where expression of the protein is very low or unknown, the use of recombinant proteins or exogenous expression in a surrogate cell line may be necessary for antibody validation. Although endogenous systems are preferred for their closer representation of in vivo conditions, heterologous strategy offers several advantages.
A multiple antibody strategy is a powerful approach to antibody validation. One of the most common methods to achieve this is to immunoprecipitate (IP) the target with one antibody and subsequently detect it by western blotting with another antibody against the same target. This provides confidence that both antibodies are binding the correct biomolecule.
An orthogonal strategy for antibody validation involves cross-referencing antibody-based results with data obtained using non-antibody-based methods. This approach is critical to verify existing antibody validation data and to identify any effects or artifacts that are directly related to the antibody in question. Providing an additional level of detail to support results generated by the other strategies outlined within this handbook, orthogonal validation often utilizes data which are available in the public domain.
While a binary approach is one of the more desirable ways to evaluate antibody specificity, binary models are not always readily available and can be time consuming or expensive to produce for the sole purpose of validating an antibody. Moreover, to assess the sensitivity of an antibody in the application and protocol being used, a complementary hallmark is required.
A binary approach is one of the best ways to evaluate antibody specificity. By testing an antibody in biologically relevant positive and negative expression systems, it is possible to confirm that it recognizes the target antigen in its native environment without cross-reacting with other biomolecules present in the sample.