Published On: Thu, Mar 10th, 2022

Understanding and Limiting Cross-Reactivity in ELISA Testing

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Antibodies show a strong affinity to specific sequences on antigens. This is what makes them a valuable tool for a wide variety of research applications that involve identifying and detecting target proteins in immunoassays. Each antibody features a specific amino acid sequence that dictates its affinity to a corresponding antigen.

Researchers can use polyclonal or monoclonal antibodies. Polyclonal antibodies are utilized more often for general applications because they are a heterogenous antibody population capable of interacting with different epitopes on the same antigen. This means polyclonal antibodies are more sensitive, as well as more robust for detecting small variations in individual isotopes of proteins. Polyclonal antibodies are often faster at capturing of target proteins and binding to the antigen, and less vulnerable to changes from pH, buffer, and storage conditions. 

What Is Cross-Reactivity in an Immunoassay?
For these reasons, polyclonal antibodies are the recommended capture antibody in sandwich ELISA tests. However, they are more susceptible to cross-reactivity due to their ability to bind with multiple epitopes and the production of different clones of plasma B cells. When an antibody deployed against one specific antigen has a competing affinity toward another antigen, it can recognize two different antigens with similar structural regions. This phenomenon can cause cross-reactivity and unexpected interactions. Cross-reactivity across species is common among human antigen-derived antibodies but is not always a negative quality. For example, cross-reactivity can allow the same antibody to be used in multiple model organism species. However, it can also create inaccurate results and prevent reproducibility.

It is important to note that the specificity of an immunoassay does not solely depend on the antibody’s binding properties, but also the antigen and reagent composition and the chosen immunoassay format. While immunoassays are extremely sensitive, certain formats may lack sufficient specificity and accuracy and cause cross-reactivity. This is more common in competitive assays but can occur with any assay if a researcher fails to follow the proper steps for identifying and addressing possible cross-reactions. The risk of cross-reactivity varies with the similarity between the protein sequence of the immunogen and the potentially cross-reactive protein sequence.

Cross-Reactivity in ELISA Testing
ELISA is widely used throughout research organizations, clinical laboratories, and pharmaceutical companies because it offers standardized quality control with simple, rapid operation. Accuracy and reproducibility are crucial for the validation of any immunoassay format but are especially important for ELISA kits. Accuracy refers to the degree of similarity between the determined value and the known true value under specific conditions. Reproducibility refers to the variation in results from samples tested within the same assay or tested in different assays (at different times or performed by different experimenters). 

Cross-reactivity in ELISA can occur through non-specific binding, or substances that adsorb to the solid phase and cause high background noise or false signals. It can also occur when an enzyme-labeled secondary antibody negatively impacts the selectivity of the assay, leading to inaccurate and irreproducible findings. This usually occurs by altering the concentration of the target analyte in the sample (over- or under-estimation) or altering antibody binding. Additionally, when detection antibodies labeled with biotin are used along with streptavidin-HRP conjugates in an indirect sandwich ELISA, they can interact unexpectedly and produce false signals, particularly in clinical immunoassays.

How to Limit Cross-Reactivity in ELISA Testing
The easiest and most straightforward way to reduce cross-reactivity is to select the appropriate antibodies for an application. Primary (capture) antibodies directly bind to the target antigen and secondary (detection) antibodies bind to the primary antibodies, allowing for signal detection and amplification. Monoclonal antibodies are ideal for use as the primary antibody, as they establish high specificity. Polyclonal antibodies should be used as the secondary antibody to maximize detection sensitivity. The secondary antibody must recognize the isotope of the primary antibody. If the primary antibody is a fragment rather than an entire antibody, the secondary antibody must be fragment-specific to decrease noise or unwanted signaling.

When deciding which antibodies to use in an experiment, examine the immunogen sequence of each antibody to determine which epitope the specific antibody will target. There are multiple online tools that allow you to input the protein sequence from this product information, align it with the protein sequence from the species you want to test, and receive a percentage of alignment. Basic Local Alignment Search Tool (BLAST) works by comparing the nucleotide or protein sequence of interest with sequences in a database and calculating the statistical significance of the match. In most cases, if these sequences overlap by 75% or more, they are likely to cross-react.

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