Demand for immunoassays in drug discovery is increasing steadily. These sensitive assays are used in both the preclinical and clinical stages of drug development, where they can evaluate drug response biomarkers, immunotherapy success, and toxicity. Immunoassays are invaluable for winnowing drug candidates in early discovery or for measuring therapeutic responses in a clinical trial — but they are also challenging to design, laborious to run, and costly in terms of time and sample use.
Multiplexing technology can address these limitations by reducing the need to run numerous assays. By testing multiple targets in a single reaction, drug discovery scientists can dramatically reduce the amount of sample and hands-on time required. This approach also allows users to generate more comprehensive data about each analyte of interest.
Even with these benefits, multiplexing assays still present some technical difficulties. While individual assays can be designed such that each component — including buffer and antibodies — is the perfect match for an individual analyte, multiplex assays have to be designed with components that work for all analytes. That often requires certain compromises, such as finding a buffer that works well enough with every analyte within a panel even though it may not be the ideal buffer for any individual analyte. When done correctly, multiplex assays yield meaningful and accurate results despite these compromises.
In drug discovery pipelines, a standard single-analyte immunoassay is typically performed with a plate-based ELISA involving a pair of antibodies to detect an antigen or another marker of interest. With multiplexing, that same reaction takes place in parallel with many other reactions, often using beads as a substrate to attach the antibodies and antigens. Color-coded beads, lasers or LEDs, and other technical elements are used to increase the number of distinct reactions that can be run simultaneously. Some versions of this technology can run hundreds of assays on hundreds of samples each day, making multiplexing a very cost-effective, automated, and rapid testing method.
Multiplex assays are also miniaturized compared to ELISAs, western blots, PCR reactions, and other standard single-analyte assays. Sample volume that might only be enough to run a single ELISA can power hundreds of assays with multiplex technology. This is particularly important in drug discovery pipelines, where samples from patients or even model organisms, such as mice, can be quite limited. With standard assays, scientists would have to prioritize which analytes are measured because there isn’t sufficient sample to run all of the desired assays; multiplexing makes it possible to perform more measurements on each sample, generating quantitative information to help in the discovery process and preserving samples for future use.
Implementing multiplex assays is not without its own difficulties, although once overcome the benefits outweigh the initial technical challenges. Designing assays to span multiple analytes involves finding the right buffer and optimal antibody pairs, and then performing validation studies.
When choosing a buffer, it is important to select a system that performs as needed on specifications such as linearity or recovery for each analyte in the panel, while managing any matrix effects. These attributes will help deliver accuracy and sensitivity in the final multiplex assay. The best buffers will meet assay performance criteria with a broad range of analytes, allowing for a high level of multiplexing.
Antibody selection is just as important. Just like standard sandwich ELISA, multiplex assays typically require antibody pairs — one to capture the analyte and another to measure it. Finding pairs that accomplish these tasks can involve extensive screening when considering performance criteria such as linearity, specificity, and sensitivity. Scientists developing these assays must also ensure that the candidate antibodies produce consistent results.
Finally, validating new assays requires biological validation as well as analysis of assay performance. Immunoblots are useful tools for validating the function of antibody pairs, allowing users to confirm specificity by showing that the protein detected by the antibodies has the expected molecular weight. The multiplex assay as a whole must also be assessed for performance; this important step determines whether the results produced are sensitive, precise, and accurate. For example, repeated testing of the same assay can reveal any inter-assay variability and potentially identify elements of the process that are not as robust as needed.
Being attentive to these factors can ensure optimal performance of multiplex immunoassays. For scientists in academia or pharma/biotech who prefer not to handle the complexities of assay design, validation, and manufacturing, outsourcing to experts with access to extensive antibody libraries, and knowledge in assay development and manufacturing can be a helpful way to get the work done while keeping internal resources focused on generating results from previously established assays. In-house assay design is an excellent option for researchers with the expertise and resources to address antibody and buffer selection and comprehensive assay validation.
Multiplex immunoassays are an effective means of lowering costs, reducing hands-on time, and limiting the amount of sample needed to generate actionable insights. Indeed, drug discovery pipelines where multiplexing has been adopted typically find that the cost reduction in reagents alone is significant. Keeping an eye on a few technical challenges will pay dividends in the accuracy, reproducibility, and specificity of results.
Andrew Hudacek is a Custom Luminex Assay Supervisor at Bio-Techne.
Filed Under: Drug Discovery