What is Cross-Reactivity?
May 13, 2022
Cross-reactivity is the most common cause of interference in immunoassays. It occurs when an analyte (cross-reactant) in a substance is very similar to the target analyte, and binds to the target antigen. The cross-reactant binding to the antigen is not always a perfect fit, but it can be close enough to result in a false positive or an overestimation of analyte concentration. It is very important to minimize cross-reactivity in immunoassays to produce an accurate result and reproducible data.
The analyte and cross-reactant both have epitopes; structural regions that are the site of recognition for antibodies precoated on the microtiter plate. The epitope has a specific amino acid sequence that determines its affinity for the binding site. If the amino acid sequence of the cross-reactant is similar to the sequence of the analyte, they will compete for the binding site on the antibody.
Cross-reactivity can invalidate experimental results and negatively influence scientific reproducibility. To prevent this, it is important to assess the percentage homology of the analyte with similar proteins that may be in the sample. You can determine percentage homology by pair-wise sequence alignment via NCBI-BLAST, or by contacting us if you have concerns. To use NCBI-BLAST, please contact us for the immune sequence of the specific ELISA kit you are interested in. A percent homology over 60% has a high chance of cross reactivity, and anything over 75% is almost guaranteed to cross-react.
Cross-reactivity may be influenced by temperature. Research has shown that cross-reactivity decreases with incubation time until an equilibrium is reached. Given that reaction rates increase with temperature, equilibrium is reached sooner at higher temperatures. This explains how increasing the incubation temperature may decrease cross-reactivity. Another piece of evidence that supports the connection between temperature and cross-reactivity is that the equilibrium constant may vary with temperature. If the change in equilibrium constant is different for the analyte and cross-reactant, then cross-reactivity would be temperature driven. Overall, the effects of temperature on cross-reactivity are unpredictable and different for every case, but there does seem to be a correlation.
The best way to achieve confidence and decrease cross-reactivity in your immunoassay performance is to choose high quality reagents. Always select antibody products with high analyte affinity and specificity. At Reddot Biotech, we are proud to put quality first and offer reliable products.