Developments in Multiplexing Aim To Reduce

Immunoassays are used for a wide variety of proteomic and genomic applications in drug discovery and development, including monitoring toxicity, detecting biomarkers of drug response, evaluating therapeutic responses in a clinical trial, and more.

Despite its prevalence, even the best professionals strive to run every immunoassay in exactly the same way. Run-to-run variability caused by minute differences between operators, product variation in buffers and reagents, or even small changes in environmental conditions in a laboratory, has been a constant challenge for drug discovery and development teams.

Balancing performance, specificity, and reproducibility in immunoassays

Part of the problem is due to the serial nature of immunoassays. Whenever results are generated from separate reactions or separate wells, there is potential for introducing confounding variables. For many applications, especially high-throughput ones, multiplexing has offered consistency and allowed scientists to significantly improve the reproducibility of their assays. However, there are still many applications where analytes must be interrogated separately, especially when issues such as cross-reactivity must be factored into the equation.

The COVID-19 pandemic has put this challenge front and center for many researchers. The measurement of antibody isotypes against the SARS-CoV-2 virus, a process that should be simple and reproducible, has been hampered by technical protocols that require the detection of these isotypes by a single reporter in separate reactions. The results of these methods are not always as comparable as scientists would like.

In an ideal scenario, antibody isotypes and other closely related analytes would be measured in the same reaction or well to avoid any potential variability inherent in separate reactions. Using a single reaction approach would also reduce sample volume requirements and experimental costs, since all data points would be produced from a single reaction.

The advantages of multiplexing over single plex methods

For years, scientists have embraced multiplex assays to generate more robust and reproducible results compared to separate single-complex immunoassays such as ELISAs. There are a few different approaches to multiplexing, but in general, multiplexed assays improve the quality of your results by testing many targets or analytes in a single reaction.1,2 Additionally, using identical experimental conditions for all the analytes you test makes make the results generated between analytes more easily comparable.

Depending on the multiplex approach used, other advantages of these higher throughput systems often include miniaturized reactions that reduce sample input requirements and reduce reagent usage. They also minimize manual labor time, allowing scientists to move on to other tasks and reducing the cost of each experiment. Importantly, they can detect hundreds of analytes in a single run.

Recently, a new feature of multiplexing technology has been developed to further reduce variability in immunoassays. The dual indicator feature, which uses two channels of information, allows scientists to collect two different measurements of each analyte in a sample. This would be especially useful when measurements come from related elements, such as detection of antibody isotypes, the presence or absence of post-translational modifications in nucleic acids or proteins, or free versus bound drug in a sample.

In a recent study, a multiplex approach was used to detect IgG and IgM antibodies against three SARS-CoV-2 virus antigens (the spike protein, the receptor-binding domain, and the nucleocapsid protein).3 The technique involved a bead based on a multiplexing system in which dual reporter channels captured two distinct signals from each individual bead, effectively duplicating the data produced from the study and ensuring that the experimental conditions for both antibody isotypes were identical. The data generated with this method provided a more accurate and comprehensive view of the immune response to the virus compared to traditional methods.

Factors to consider when developing a multiplex assay

With various options for multiplexing immunoassays, there are many key factors to consider when selecting the best option for your lab or experiment: