How do I know if the antibody will cross-react?

Cross-reactivity occurs when an antibody raised against one specific antigen recognizes two antigens that have similar structural regions.

What is cross-reactivity?

An antibody has a specific amino acid sequence (the Fab region) that dictates its affinity for a specific antigen. Cross-reactivity between antigens occurs when an antibody raised against one specific antigen has a competing high affinity toward a different antigen. This is often the case when two antigens have similar structural regions that the antibody recognizes.

Cross-reactivity can invalidate the results of an experiment and thereby impact scientific reproducibility. Thus, testing an antibody for cross-reactivity with closely related proteins is a critical validation experiment.

How do I check cross-reactivity?

Cross-reactivity is not always a negative quality. It can sometimes improve an antibody’s utility. For instance, cross-reactivity of an antibody for a target across species allows the same antibody to be used in multiple model organisms.

Cross-reactivity across species often occurs for human antigen-derived antibodies. Many human antibodies in the Proteintech catalog have significant cross-reactivity with the homologous proteins in non-human models, such as mouse, rat, monkey, or zebrafish.

Based on the homology of a protein sequence and publications using our antibodies in non-human organisms, many of Proteintech’s antibodies can be used not only for human samples, but also for a variety of organisms. Homology can be assayed using a pair-wise sequence alignment of immunogen and target antigen through the NCBI-BLASTwebsite, and further information can be checked at the Universal Protein resource website UniProt. 

What is the “right number” for homology to predict cross-reactivity?

Based on our experience, if the immunogen and the sequence of the potential cross-reactive protein share 75% sequence homology, it is predicted to cross-react. If your sample is non-human, a polyclonal is recommended because there is a mixture of epitopes recognized.

Does my antibody cross-react with other species?

It depends on the extent of protein sequence similarity between the immunogen and the potential cross-reactive protein sequence.

How to avoid cross-reactivity in western blot

If possible, it is recommended to use the secondary antibody specific to the first antibody used; for example, if the primary antibody is mouse, then the secondary antibody should be anti-mouse IgG-HRP. In case the secondary antibody is polyclonal and generated against the whole IgG molecule, then rabbit antibody recognizing the primary mouse Ab might not be the best choice because of the recognition of the common Kappa Light Chain region.

How to avoid cross-reactivity during immunostaining experiments

Cross-reactivity might occur in immunostaining assays using tissue-derived samples; therefore, it is common to use fragments, rather than entire IgGs, as primary immunoglobulins (for example, while working with mouse antibodies on mouse tissues). Additionaly, while doing a multiple staining, to ensure that there is no cross-reaction, try to use different secondary antibodies, for example, 568 rabbit anti-goat with a 488 rabbit anti-mouse, which are quite common and easy to find.

What is the secondary antibody cross-adsorption and cross-reactivity?

Cross-adsorbed secondary antibodies are those polyclonal antibodies that are manufactured with an additional purification step in order to filter out members that bind to off-target species of immunoglobulin (IgG). This process can decrease species cross-reactivity and increases specificity. Depending on the experiment set up there are cross-adsorbed and highly cross-adsorbed secondary antibody varieties. 


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