Many articles discuss validation, and most of them address the antibody’s specificity. But there is still confusion, especially when specificity of an antibody is thoroughly confirmed and later on it appears the antibody cross-reacts to other proteins. We will try to create some clarity on these issues.
First some definitions:
Specificity: This is defined by the epitope the antibody binds to.
When specificity is low, the antibody would bind to several different epitopes. Some of these epitopes may be similar to epitopes owned by non-related other proteins, thus causing detection of these proteins. Specificity increases when the antibody (at a given dilution) binds to epitopes closely related to the specific epitope the antibody was generated to. The highest specificity is reached when the antibody binds to no other epitope but one.
From the above comments it follows that a monoclonal antibody, having one epitope to bind to, is inherently specific. However, when the epitope is NOT unique, the antibody will bind to any other protein having this same epitope. This does not make the antibody less specific.
From the above comments it follows that a polyclonal antibody is inherently not specific to one single epitope. To approach the mono-specificity of monoclonal antibodies, polyclonal antibodies can be generated to small peptides, so to limit the variation of epitopes within the length of the immunizing peptide. The sequence of the immunizing peptide contributes to the specificity of the polyclonal antibody (when this antibody was affinity purified using the immunizing peptide).
Selectivity: This is defined by the antibody's binding to a unique epitope.
When selectivity is low, the epitope is shared with other, related, proteins. Selectivity increases when the epitope is unique for the protein it is intended to detect, while other related proteins present will not be detected.
From the above comments it follows that a monoclonal antibody may bind to a specific epitope, but when this epitope is NOT unique, its specificity will not prevent cross-reactivity to other related proteins.
From the above comments it follows that a peptide-generated polyclonal antibody can be highly selective when the sequence of the immunizing peptide is unique, so that even a mixture of epitopes within this peptide will retain the antibody’s ability to be selective.
We should note that a polyclonal antibody generated and affinity purified using an entire protein, by definition will be a mixture of antibodies binding to multiple epitopes, but it can still be used as highly selective when diluted strongly enough to detect only those in the mix with the strongest signals to unique epitopes. Thus its use can be deemed specific and selective in certain applications.
How is specificity tested?
Too often we see an antibody only tested to its intended target. Get a signal, and claim that the antibody works. This approach is often used in ELISA and in WB on recombinant target protein. However, when this protein was in buffer (without other proteins around), such approach is not sufficient to demonstrate specificity.
The appropriate testing of specificity is to have real biological material analysed and to compare in the presence (preferably at natural levels) and in the absence of the intended target protein. Ideally, the signal responds proportionately with the amount of target present in the biological material. When the biological material does not show any significant levels of non-specific signals (at the optimal dilution of the antibodies), the antibody can be deemed specific.
How is selectivity tested?
The testing on selectivity is harder to find across catalogues. It requires testing cross-reactivity to closely related other proteins compared with the intended target protein. When such cross-reactivity cannot be detected, while giving a strong signal of the intended target at the same time and at the same antibody dilution, the antibody is deemed selective.
This is important when biological material is used that potentially possess a mixture of different closely related proteins. For example, breast tissue may have HER1, HER2, HER3 and HER4 expressed. Any HER2 antibody used to diagnose HER2-type breast cancer, may or may not cross-react with the other HER receptors. When they do, such antibodies are not selective to HER2 and false positives will be found.
Aeonian Biotech now offers antibodies with both evidence of specificity and evidence of selectivity. In addition our products show molecular integrity data and evidence of fitness for at least one application.