The benefits of multiplex immunoassays seem rather obvious—with them, you can do more, faster. And while that is true, their advantages are more wide-ranging than just higher throughput.

Biocompare recently asked two experts—James Murray, Director of Immunoassay Development at Abcam, and Candice Cox, Global Marketing Manager, Immunoassays, Protein Quantitation Business at Bio-Rad Laboratories—to discuss multiplex immunoassays with us. We wanted to know why researchers should be using multiplex immunoassays now. They answered that question and more—also addressing ideal applications, providing tips on best practices, and sharing insights on novel products and technologies that are continuing to improve upon multiplex immunoassays. All valuable information that can benefit your research. Check out our conversations below.

BC: What are some of the advantages of using multiplex immunoassays?

James: One main advantage to using multiplex immunoassays is their high-throughput potential, providing more results per sample, with lower sample volumes (i.e., miniaturization), which translates to a lower price-per-data point compared to traditional singleplex immunoassays (e.g., ELISAs). The measurement of multiple analytes in a single sample also gives a “total picture” snapshot with more reliable data, as it eliminates the need to compare data captured at different time points and from different experiments, which makes these traditional methods more prone to user errors.

Candice: In comparison to other technologies likes ELISAs, multiplex immunoassays allow users to maximize data obtained from a minimum volume of sample. For many ELISAs, it requires 100 microliters or more to get one answer for one specific analyte, but with multiplexing, depending on how large a panel is, you are able to get a lot of information from very minimal samples (somewhere around 10–13 microliters, most of the time). A lot of researchers who work with animal models like mice—from which you get very low sample volume to begin with—really appreciate the fact that you can get so much data from such a small sample volume.

Multiplexing also increases throughput by allowing you to save some time. Many available immunoassays will require an incubation of anywhere between a couple of hours to overnight. For Bio-Plex assays, the primary incubation is between 30 minutes and an hour, and, overall, you can get data in as little as three to four hours. That is a big advantage for people who want data quickly.

Also, the fact that you can get all these answers for many different analytes at the same time allows for more consistent data. And because you do not add confounding variables like different operators or different buffer systems, you really get a snapshot of the profile you are looking at, whether it be cytokines or other proteins.

BC: For which applications are multiplex assays ideal?

James: Multiplex immunoassays, including Abcam’s FirePlex immunoassays with optimized matched antibody pairs, are best used for measuring targets in biofluids, including circulating biomarkers and signaling pathway molecules like hormones and cytokines. For high-throughput biomarker discovery, immune cytokine profiling, and studies with very large patient cohorts, FirePlex-HT offers the same high standards of performance and multiplexing found in our original FirePlex immunoassays, but in a high-throughput, automation-friendly format.

 Figure 1. Example of Human Discovery Cytokines Panel 1 standard curves. Mean background and control particle-subtracted data values are graphed (+/– standard deviation). Image courtesy of Abcam.

Candice: Multiplex immunoassays are ideal for anything with limited precious sample, like CSF, mouse samples, neonatal samples, and samples that need to be spread over multiple tests. Our Bio-Plex kits cover multiple disease states, including cancer research, immunotherapy, diabetes, cardiovascular disease, autoimmune and infectious diseases, and many more. So it really just depends on what you are doing and what type of answer you require.

BC: Can you provide some best practices/tips for designing effective multiplex immunoassays?

James: An issue we frequently come across is data that relies on the results of a single sample, which often negatively affects the robustness of statistical analyses. To avoid this issue, we recommend that researchers always set up replicate samples in their experiments instead of using repeat measurements.

In addition, the immunoassays you use should be well-validated. Off-the-shelf assays such as our FirePlex immunoassays have undergone significant validation testing with suitable negative and positive controls, but if you are putting together your own multiplex assay, there are multiple steps that are important to achieving a high standard of assay validation: (1) specificity, including the detection of any antibody cross-reactivity or interference (from both protein and antibody); (2) sensitivity; (3) dynamic range; (4) linearity between standards and samples; and (5) spike recovery in complex matrices such as biofluids.

Figure 2. Example data set of various human biological sample types from normal healthy donors. Each biological sample was measured at two dilutions (1:4 and 1:10) in duplicate. The concentrations of each cytokine were interpolated from their respective standard curves and corrected for sample dilution. The mean interpolated dilution factor corrected values are graphed. Image courtesy of Abcam.

Candice: When designing your own multiplex immunoassays, make sure to use highly specific antibodies, otherwise the results you get will not be meaningful. And when assessing specificity, make sure to carry out cross-reactivity testing against specific antigens and against detection antibodies.

Make sure your results are reproducible by evaluating well-to-well and plate-to-plate variability. Testing things like linearity and parallelism during assay development is also a good practice to ensure you are getting accurate results with the suggested sample dilution factor and with various sample types. You will also want to verify that the matrix for the kit-provided diluents behaves similarly to whatever biological fluids you are analyzing with the kit.

You will also need to ensure that you have a large working assay range, allowing you to look at normal healthy samples as well as disease samples. Your analytes will often have a wide range of concentrations, with some proteins being overexpressed and others underexpressed. Having a nice working range will allow you to measure each of your proteins of interest in their specific biological state.

Also, just in terms of running the assay, we recommend that you keep consistent incubation times as recommended by the vendor to get plate-to-plate consistency and quality data. Minimize the number of freeze-thaw cycles your sample goes through because these can be hard on proteins. Finally, use a multichannel pipette so that you can get everything onto the plate efficiently at each step. With Bio-Plex assays having an incubation time as low as 30 minutes, getting everything on the plate quickly is important to ensure consistency in the incubation times from one end of the plate to the other.

BC: Are there any advances on the horizon to make it easier to use multiplex immunoassays?

James: Miniaturization and easier, simplified workflows can make data more reliable and save sample for other assays or for additional replicates. In addition, recent improvements in sensitivity and the field of “multiomics”—which is the measurement of more than one class of molecule, for example, both miRNA and proteins—are revolutionizing our view of biological systems. Both of these advances can be achieved with Abcam’s FirePlex platform, including miRNA assays and immunoassays.

Candice: It has been a long time since multiplex immunoassays moved from nonmagnetic to magnetic beads, but automating the washes really made it so that you wouldn't have to be an expert or a frequent user to get good, reproducible results. Now, you basically just have a magnet holding down the beads and you can automate the wash. This results in little residual volume, which helps to increase your precision.

Software that allows users to assess their assay performance and look at the quality of the results is also important. Many customers have said that our Bio-Plex Manager and Data Pro software makes it easier to QC and analyze the large amount of data generated and also helps them to make biological sense of their data.

There are also a few companies that are automating a larger part of the workflow, which increases throughput and precision.