How To Determine Antibody Cross-Reactivity

Choosing the right antibody can get complicated, so it’s not surprising that customers frequently ask us questions like, “If this antibody is validated for human, will it cross-react with rat, too?”

First, let’s review the concept of cross-reactivity. Cross-reactivity between antigens occurs when an antibody targeted against one specific antigen is successful in binding with another, different antigen. The two antigens in question have similar three-dimensional structural regions, known as epitopes, which allow the antibody for one antigen to recognize a second antigen as being structurally the same.

Antibody cross-reactivity figure

In order to determine if an antibody validated for human will cross-react with rat, we should take a look at the immunogen sequence (aka antigen sequence) of the antibody. At Boster Bio, we provide the immunogen sequence on our antibodies’ product pages. The immunogen sequence will tell you information about which epitope the antibody will target.

You may be wondering, how do I do that? The first step is to copy the provided sequence information and align the sequence with the protein sequence from the species you would like to test. In order to compare the sequence alignment between two species, there are websites which provide tools for calculating the percentage alignment. One commonly used tool is called the Basic Local Alignment Search Tool (BLAST). This program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. It allows you to enter your immunogen sequence of interest, and select the species you want to compare with. We recommend an alignment score of over 85% as a good indication that an antibody may cross-react. For example, if the homology of the human sequence and rat sequence is higher than 90%, then, in theory, the antibody will cross-react with rat.

The following image shows an example of what the results of a sequence alignment might look like. In this example, there is a 100% identical match for the sequence of the Gap Junction Protein in both sheep and human. This indicates the antibody will most likely cross-react with both species.

Blast alignment example

There are many variables which could affect whether an antibody will bind in another species. For example, if the antibody is a phosphorylated antibody, cross-reaction with another species may not occur when the protein in the sample does not have the same phosphorylated epitopes. Therefore, even if the sequence alignment is high, that does not guarantee how the antibody will actually perform in experiments.

Now that you understand the concept of cross-reactivity and sequence homology, you can better determine whether an antibody may detect in an untested species. If you have any further questions, don’t hesitate to contact the Boster Support Team for more information.

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