Antibodies long had the reputation of a magic bullet, and thousands of researchers still use them every day to identify and isolate proteins in a large variety of applications. But in the last few years, the fame of antibodies has become tarnished: cross-reactivity and variability of the antibodies are the main culprits. The "Reproducibility Crisis" or "Antibody Crisis" are two names for this phenomenon.
We at Synaptic Systems aim to characterize and validate our products as best as possible to provide antibodies of highest quality and reliability. All our antibodies are produced in-house and therefore we have full control over batch testing and quality control. To date nearly one fifth of our antibodies are Knock-out or Knock-down validated. We strive to raise this number, so if you have a Knock-out model for one of our antibodies which has not been tested yet, please contact us at firstname.lastname@example.org.
The quality of an antibody-based reagent mainly depends on two major properties: affinity and specificity.
Affinity measures the strength of an interaction between an epitope and an antibody's antigen binding site. The higher the affinity of an antibody, the higher the sensitivity of an antibody-based assay. Specificity measures the degree to which an antibody differentiates between different antigens. This implies that even the best antibodies have a detection limit or may show false positive results in extreme situations. If, for example, the target antigen of a highly specific and sensitive antibody is very rare in the sample to be analyzed, the sample amount is often increased and the antibody is used at higher concentration to amplify the signal. If another target is present in the sample that is weakly bound by this antibody, the unfavorably high concentrations of this off-target and the antibody itself can then result in unspecific binding and false positive results.
Different experimental approaches require different antibody properties since the antigen is presented in different ways. The antigen may be a rather linear stretch of amino-acids in western blot detection after denaturing SDS-PAGE, or a more or less native structure in immunoisolation of cells, immunoprecipitation, or ELISA-based approaches. In immunostainings, cross-linked, chemically modified structures in fixed cells or tissues have to be detected. This means that one antibody may show excellent performance for one application but completely fail in another. This also means that specificity in one assay does not guarantee specificity in a different application.
Validation has to be carried out for all applications independently.
What validation methods are available and what do they tell us?
Omitting the primary antibody
This is a useful and important control to test for potential background caused by the secondary reagent. However, this experiment gives no information about the quality of the primary antibody.
Signal of the antibody is in accordance with literature data
The observed molecular weight in WB, the tissue distribution, and the staining pattern are consistent with the literature. This validation method is easy to perform but has also some restrictions; e.g. the right molecular weight of a band in WB does not necessarily tell you that the band is the desired target protein. There are easily hundreds of proteins running at the same velocity in a WB that can lead to misinterpretation of the result. The data in the literature can also be incorrect or incomplete.
Blocking/pre-adsorption of the antibody with the immunogen
Especially for polyclonal serum, this is a useful experiment to determine if an observed signal is related to the immunized antigen. If a signal disappears after pre-adsorption, the signal has a high probability of being specific. However, the possibility of cross-reactivity to other proteins sharing a similar antibody binding epitope cannot be excluded (suggested protocol for pre-adsorption can be found here).