Antibody cross reactivity and how to avoid it

What is antibody cross reactivity

Antibody cross-reactivity is the ability of an antibody to bind to multiple antigens or epitopes that are structurally similar or related in some other way. This can occur because the binding site on the antibody, known as the paratope, can recognize and bind to more than one epitope on different antigens. This can be a useful property in some cases, as it allows an antibody to recognize and bind to multiple related antigens. However, it can also be a disadvantage if the cross-reactivity causes the antibody to bind to non-specific targets, leading to false positives or other errors in the assay. Therefore, it is important to carefully evaluate the degree of cross-reactivity of an antibody to ensure that it is specific enough for the intended use

How to determine it

There are several tests that can be performed to determine if an antibody has any cross-reactivity with another antigens. These tests include:

• Immunoblotting (Western blot): Antigen is separated by electrophoresis and then transferred to a membrane. The membrane is then incubated with the test antibody and any bound antibody is detected using a secondary antibody that is linked to an enzyme that produces a visible signal. The presence of a signal indicates that the test antibody has bound to the target antigen, and the size of the signal can provide information about the specificity of the binding.
• Immunoprecipitation: Antigen is incubated with the test antibody, and any bound antibody-antigen complexes are precipitated out of solution using a chemical or physical process. The precipitated complexes are then analysed to determine if they contained the target antigen, which would indicate that the test antibody has cross-reactivity with that antigen.
• Competitive ELISA: Antigen is incubated with the test antibody and a known quantity of a reference antigen that is similar to the target antigen. The presence of the reference antigen competes with the target antigen for binding to the test antibody, so the amount of target antigen bound to the antibody is inversely proportional to the amount of reference antigen present. By comparing the amount of bound target antigen in the sample to a standard curve, it is possible to determine the amount of target antigen in the sample and therefore the degree of cross-reactivity with the reference antigen.
• Antibody array: Antigen is incubated with the test antibody, and any bound antibody is detected using a microarray of antibodies that are specific to a wide range of different antigens. The presence of a signal for a particular antigen on the array indicates that the test antibody has cross-reactivity with that antigen.

By performing one or more of these tests, it is possible to determine the degree of cross-reactivity of an antibody with other antigens.

Different factors affecting it

There are several factors that can affect the degree of cross-reactivity of an antibody, the most common factors will be discussed below:

1. The amino acid sequence of the antigenic epitope: The more similar the amino acid sequence of the epitope is between two antigens, the more likely it is that the antibody will bind to both antigens.
2. The three-dimensional structure of the antigenic epitope: Even if the amino acid sequence of the epitope is not similar between two antigens, the antibody may still bind to both if the epitope if it has a similar three-dimensional structure.
3. The affinity of the antibody for the antigen: If the affinity of the antibody for the antigen is high, it is more likely to bind to the antigen even if the epitope is only slightly similar to the target epitope.
4. The concentration of the antigen in the sample: At low concentrations of the antigen, the antibody may have a higher likelihood of binding to non-specific targets due to the increased availability of binding sites on the antibody.
5. The conditions of the assay: The pH, temperature, and other conditions of the assay can affect the binding of the antibody to the antigen and can therefore influence the degree of cross-reactivity.

By understanding these factors, it is possible to optimize the assay conditions and choose the appropriate antibody to minimize cross-reactivity and maximize specificity.

How to avoid it

There are several steps that can be taken to avoid or minimize antibody cross-reactivity:

• Choose an antibody that has been specifically designed and validated for the target antigen of interest. This will ensure that the antibody has high specificity for the target antigen and is less likely to bind to non-specific targets.
• Use immunoblotting or immunoprecipitation to confirm that the antibody binds specifically to the target antigen, and not to non-specific targets.
• Perform a competitive ELISA to determine the degree of cross-reactivity of the antibody with structurally related antigens. This can provide valuable information about the specificity of the antibody and help to identify potential sources of cross-reactivity.
• Optimize the assay conditions to maximize the specificity of the antibody. This may involve adjusting the pH, temperature, or other conditions of the assay to promote specific binding of the antibody to the target antigen and reduce non-specific binding.
• Use multiple antibodies that recognize different epitopes on the target antigen. This can help to reduce the likelihood of cross-reactivity, as each antibody will bind to a different epitope and therefore have a lower chance of binding to non-specific targets.

By following these steps, it is possible to avoid or minimize antibody cross-reactivity and ensure that the assay results are specific and reliable.

Why it is important to have no antibody cross reactivity

It is important to have zero or minimal antibody cross-reactivity in an assay because cross-reactivity can lead to false positives or other errors in the assay results. For example, if an antibody has cross-reactivity with a non-specific target, this can result in a false positive signal. In this case, the results obtained will display an overestimation of the amount of target antigen present in the sample. This can have serious consequences in medical or other critical applications. Therefore, it is essential to minimize or eliminate cross-reactivity in an assay to ensure that the results are specific and reliable.

Final thoughts

It is important to know the degree of cross-reactivity of an antibody because this information can help to ensure that the assay results are more specific, accurate and reliable. It is also vital to design the assay in a way that minimizes or eliminates the potential for any false positive or other errors occurring. The results obtained should be correct since these procedures are used to make critical decision for frontline diagnostic clinical applications.

Related Pages

1. ELISA Principle
2. ELISA Protocol
3. ELISA Sample Preparation
4. Analysing ELISA Data
5. General ELISA FAQ
6. ELISA Trouble Shooting FAQ
7. Comparison Between ELISA And EIA
8. Different ELISA Detection Strategies