Difference between antibody specificity and selectivity

Introduction

Many articles discuss antibody validation. However, most articles only address specificity without properly addressing selectivity. Such omission leads to confusion when antibody specificity was thoroughly confirmed, yet later on, it appears the antibody cross-reacts to other proteins. This blog creates clarity about the difference between specific antibodies and selective antibodies.

Definitions

Antibody specificity: This is defined by the epitope the antibody binds to.

When specificity is low, the antibody binds to several different epitopes. Certain epitopes displayed by non-related (off-target) proteins can be similar to the target epitopes, thus causing cross-reactivity. For a high specificity, the affinity (that is the strength of binding) to the target protein must be much higher than to other proteins. The difference between those affinities decrease with lower antibody specificity. Specificity increases when the antibody (at a given dilution) binds to epitopes closely related to the specific epitope of the target protein. The highest specificity is reached when the antibody binds to no other but one defined epitope.

This definition of specificity leads to the following observations:

• A monoclonal antibody, binding to a single epitope, is inherently specific. However, when the epitope is NOT unique, the antibody will bind to any other protein exposing this same epitope. This cross-reactivity does not make the antibody less specific.
• A polyclonal antibody is inherently not specific to one single epitope. To approach the high specificity of monoclonal antibodies, polyclonal antibodies can be generated to small peptides. This entails that the variation of epitopes within the length of the immunising peptide is limited. The unique sequence of the immunising peptide contributes to the specificity of the polyclonal antibody, especially when this antibody was affinity purified with the use of the immunising peptide.

Antibody selectivity: This is defined by the antibody binding to a unique epitope.

When selectivity is low, the epitope is shared with other proteins. Selectivity increases when the occurrence of the epitope across the different proteins is rare. Selectivity is maximal when the epitope is only present on the target protein. A selective antibody does not cross-react with any off-target protein that is present in the assay and thus it exclusively binds to the designated target protein.

This definition of selectivity leads to the following observations:

• A selective monoclonal antibody binds to a unique epitope, and therefore it does not cross-react to other (related) proteins.
• A peptide-generated polyclonal antibody can be highly selective when the sequence of the immunising peptide is unique, such that even a mixture of epitopes within this peptide will retain the antibody’s ability to exclusively bind to its designated target protein.
• We should note that a polyclonal antibody generated and affinity-purified using an entire protein, is by definition a mixture of antibodies binding to different epitopes. Such antibody can still be highly selective when thoroughly diluted, ensuring that only the strongest signals of unique epitopes are detected. Thus, its use can only be deemed selective in certain applications and under certain experimental conditions.

Antibody integrity

The principles discussed in this blog only apply when the antibody is fully intact, and its molecular integrity is not compromised. The antibody’s integrity can be compromised due to the below listed variables, which may also cause the loss of specificity and selectivity:

There is also a loss of specificity when the hybridoma-derived monoclonal antibody contains a mixture of different immunoglobulin chains. Some hybridomas express an extra light chain (from the cell fusion partner), or an extra heavy chain (from a fusion with two different B-cells).

A separate blog with further details on antibody integrity is available, but this blog is only accessible to members.

We publish many of our product pages with data of Coomassie Blue-stained SDS-PAGE showing the molecular integrity of the antibody under reduced and non-reduced conditions.

In practice

How is antibody specificity tested?

Too often we see an antibody only tested to its intended target. When a signal is obtained, the antibody is claimed to work. This approach is often used in ELISA and in WB on recombinant target protein. However, such approach is not sufficient to demonstrate specificity, because even a non-specific antibody at high enough titre would bind to this protein.

The appropriate testing of specificity is to compare real biological material with high expression, low expression, 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 is deemed specific.

How is antibody selectivity tested?

The testing of selectivity is harder to find across catalogues. Selectivity is tested by comparing the reactivity to the intended target protein with closely related other proteins. When cross-reactivity is absent (with the presence of related proteins proven by positive control antibodies), while giving a strong signal of the target protein 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 expresses a mixture of different closely related proteins. For example, breast tissue may express the combination of HER1, HER2, HER3 and HER4. Particularly to diagnose HER2-type breast cancer, some HER2 antibodies may or may not cross-react with the other HER receptors. When they do, such antibodies are not selective to HER2, and potentially lead to false positives.

Aeonian Biotech now offers antibodies with both evidence of specificity and selectivity. In addition, our products show molecular integrity data and evidence of fitness for at least one application.

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