Tracking multiple targets in one reaction is an increasing requirement of assays being used in molecular biology and medicine. Optimally, scientists want to analyze as many biological or molecular markers as possible in the same experiment to acquire better comparisons of shifts in the patterns of different targets associated with changes in different biological processes. Such multiplexing saves time and sample volumes, some of which can be precious and difficult to obtain. Tracking these molecules often involves antibody-based chemistries. Add those two things together, and you get multiplex immunoassays.
Many applications can benefit from multiplexing technologies. According to Scott Van Arsdell, vice president of immunoassay technology at Aushon Biosystems, the major applications of multiplex immunoassays include clinical trials, toxicity testing, translational research, therapeutic monitoring and diagnosis of complex diseases.
Dandan Shan, senior scientist at Quanterix, adds, “The capabilities of the multiplex technology are particularly well-suited for pharmacodynamics and pharmacokinetic characterizations of candidate drug therapies, because they allow researchers to directly quantify the biochemical impact of a drug.”
It is not uncommon for multiplex assays to provide a hundred times more data.
Reducing the amount of sample needed to analyze multiple targets can be crucial. “Many important biological specimens are available in exceedingly small quantities, including cerebrospinal fluid, exosomes, and biofluids from rodent models,” says Dan Pregibon, general manager of platform innovation at Abcam. “Compared to singleplex immunoassays, it is not uncommon for multiplex assays to provide a hundred times more data.”
A variety of techniques and products make it easier for scientists to use multiplex immunoassays, and get valuable results. As Stephen Angeloni, senior field application scientist at Luminex, explains, “Data is more accurate as all analytes are measured in the same reaction versus different reactions.”
Excellent applications
Many biological and all clinical tests require validated biomarkers. In many cases, 10–50 candidate biomarkers go into further evaluation, which can generate hundreds of thousands of samples to analyze. “Because traditional approaches like ELISA and Western blot can be laborious and inherently limited in throughput, validation studies are best served with highly automatable, multiplexed assays,” Pregibon explains. “In such cases, a multiplex assay like Abcam’s FirePlex platform allows researchers to profile 10 to 20 proteins or 20 to 50 microRNAs from only 50 microliters of biofluid, and do this over hundreds of samples.” In some cases, it takes multiple biomarkers to diagnose a disease or to assess the efficacy of a treatment. That makes throughput even more crucial.
Some of these platforms have already been used in many applications. The Luminex platform, according to Angeloni, has been used in vaccine studies, studying the development or progression of a number of diseases, treatment responses, the development of antibodies for research or as therapeutic compounds and effectiveness of different therapeutic compounds. Part of the benefit of this technology arises from its ability to measure as many as 500 targets in a 50–100 microliter sample. Moreover, this technology saves a lot of sample. “It is not unusual to be able to dilute a sample hundreds to thousands of times and still have better sensitivity than a singleplex assay,” Angeloni notes.
Moving from more traditional technology to multiplex immunoassays can make a few key improvements. For example, Van Arsdell says, “Running Aushon’s 10-plex cytokine panel enables the researcher to replace 10 separate ELISA assays in the same amount of time, using only 50 microliters total of sample volume for all 10 biomarkers—versus running 10 separate ELISA plates and consuming as much as 1,000 microliters of sample volume to acquire the same amount of data.”
Some multiplex-immunoassay platforms focus on fairly specific targets. “The ImmunoSpot multiplex platform is immune monitoring for the detection and establishing frequencies of antigen-specific B cells and T cells,” says Paul Lehmann, CEO of ImmunoSpot. This system can detect a wide range of these cells. As an example, Lehmann says, “In multiplex B cell ImmunoSpot assays, individual B cells producing antibodies of any immune globulin classes—IgG, IgM, IgA and IgE—or any subclass—IgG1, IgG2, IgG3 and IgG4—can be detected simultaneously while testing as few as 100,000 cells in a single assay.”
Many clinical applications really benefit from multiplexing. As Shan says, “Our new six-plex cytokine immunoassay can quantitatively measure a set of the most important biomarkers in inflammation, immune system regulation as well as immune-oncology.” She also notes that it can measure “clinically relevant concentrations of immunological mediators, exosomes, and microRNA.”
Beyond using small sample sizes, researchers often use multiplex immunoassays to look for small differences, such as post-translational modifications in proteins. “For example, neurological and oncology research often target intricate signal-transduction pathways where these small molecular weight modifications can have significant downstream effects,” says Taylor R. Murphy, director of technical product specialists at Azure Biosystems. “Modern investigators need to go beyond simply quantifying total protein expression and can use multiplex immunoblots to investigate the activity state—phosphorylation—or potential subcellular localization—palmitoylation—of the protein in question.” Here, scientists should use antibodies designed to target the modified protein. “Using the specificity of primary antibodies, researchers can now investigate in more detail the potential behavior of the protein of interest including half-life, activity state or other potential protein-protein interactions,” Murphy explains.
Although multiplexing aims primarily to get more from less, Lehmann notes, “There is growing need to expand the technology even further by measuring not only multiple analytes present in a sample, but also the per cell output of each analyte.” He adds, “In particular, the ImmunoSpot multiplex platform can measure a single cell producing multiple analytes—termed polyfunctionality.” Such measurements can be used to develop advanced cancer treatments, such as checkpoint inhibitors, chimeric antigen receptor T cell therapy, and adoptive cell transfers.
Troubleshooting tips
If something goes wrong in a multiplex immunoassay, the first suspects are the antibodies. “The affinity, and more importantly, specificity of each antibody used in a multiplex assay can have a major impact on overall assay performance,” says Pregibon. In all cases, scientists must look for validated antibodies developed for the specific applications. Furthermore, scientists should run positive and negative controls for every application.
Also, start small. “Before you start multiplex immunoassay development, make sure that your individual assays are working,” Shan says. “Selection of antibodies, diluents, and incubation steps can impact the performance of immunoassays.”
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In some cases, troubleshooting is platform-specific. “If you are using a Luminex 200 or FLEXMAP3D and are experiencing certain types of bead loss issues, check that the double-discriminator gating is properly set for the type of beads being used,” Angeloni suggests. “If these are not the cause of the problem, we would then start to review the types of samples being used and how the assay reactions were set up and performed to see if the bead loss is associated with sample characteristics or how the assay was processed.”
Using multiple antibodies creates the risk of cross-reactivity in some situations and can be a source of error. As Van Arsdell explains, “Multiplexing adds one additional complexity as compared to traditional ELISAs, which is cross reactivity between the analytes being tested, but this is part of the development and validation of multiplex assay kits supplied by Aushon.”
The detection method also comes into play. “When using fluorescent detection chemistries, there are some considerations to be made to reduce background noise,” Murphy says. “Protein-based blocking buffers can autofluoresce if not thoroughly washed, and detergents like tween or triton can also add to potential background.” He adds that “specialized block/wash buffers and membranes can significantly reduce background noise and improve performance.”
So, multiplexing immunoassays multiply the potential applications, but the complexity increases, as well. Buying the right technology for the task and troubleshooting as needed makes all of the difference.
Caption: Luminex beads make up a key ingredient of many multiplex immunoassays. Image courtesy of Luminex.