Developing Multiplexed Immunoassays

Multiplexed immunoassays are increasingly popular for the breadth of information they provide. Yet developing a multiplexed immunoassay is a challenging task, and is often best outsourced to save time, money, and sample material.

A growing trend toward multiplexing

Because single analyte immunoassays such as traditional ELISA have been around for decades, they currently remain more common than multiplexed immunoassays for scientific research. But this looks set to change according to Megan Larson, Senior Product Manager at Bio-Techne. “The multiplex immunoassay market continues to grow rapidly, as scientists seek more comprehensive pictures of their biological samples,” she says. “Ultimately, multiplexing allows one to save on sample, while discovering more.” As a consequence, many different types of multiplexed immunoassays have now been developed.

Many available options

According to CJ Xia, VP of Marketing and Sales at Boster Bio, multiplexed immunoassays can be broadly categorized as either surface-based or bead-based. “Surface-based multiplexing involves coating different antibodies onto a solid support, such as a microplate or nitrocellulose membrane,” he explains. “The bound analytes are then differentiated by spatial separation or by using a panel of detection antibodies, each conjugated to a different fluorophore, oligonucleotide, or other label. Bead-based immunoassays instead use antibody-coated beads for analyte capture, whereby the beads are barcoded to enable their detection with a flow cytometry like system.”

Luminex Corporation’s xMAP^® Technology is probably the best-known example of the latter. Following analyte capture, and the addition of biotinylated target-specific antibodies and PE-conjugated streptavidin, the samples are read on a dual-laser instrument, where one laser identifies the beads and the other measures the PE-derived signal. “xMAP is a proven technology for clinical diagnostics, life science research, and drug discovery labs, with over 50,000 publications citing its use,” says Dominic Andrada, Luminex’ Scientific Content Senior Marketing Manager. “With xMAP, it is currently possible to detect as many as 500 analytes in solution, giving researchers a better understanding of what is going on at different levels—from the molecular and cellular levels through to tissue, organ, and disease state.”

In addition to surface-based and bead-based multiplexing, techniques such as flow cytometry and IHC are also frequently multiplexed. “Multiplex IHC is being used more and more for discovery and translational research, but has yet to be fully adopted in the clinic, where singleplex chromogenic IHC currently is still the standard,” reports Sarah R. Klein, Senior Group Leader, Multiplex Assays Development at Cell Signaling Technology. “However, scientists and clinicians alike agree that detection of multiple biomarkers at once may better stratify patients and predict therapeutic outcomes. In the future, we will continue to see more advanced multiplexed immunoassays, enabling investigators to glean more information from precious human samples in order to better guide clinical decisions.”

Factors to consider for platform selection

When it comes to deciding which multiplexed immunoassay platform to use, researchers must consider several important factors. These include assay sensitivity and specificity, as well as sample type, sample number, and how many different analytes will be measured. “An immunoassay is only as good as its antibodies,” cautions Klein. “Without confirming the quality and validation standards of antibody reagents, researchers cannot be guaranteed reliable, reproducible results. Also, because sample preparation influences antibody performance, it is imperative to select antibodies that have been shown to perform appropriately with the intended sample material.”

Andrada agrees, adding that while the sensitivity of multiplexed immunoassays has dramatically improved in recent years (it is now possible to detect sub-picogram analyte concentrations), specificity remains paramount. “The specificity of an assay is critical to the accuracy of multiplex data,” he says. “Understanding whether the different target analytes are compatible, or if they may interfere when multiplexed, is key to avoiding cross-reactivities and false positive results.”

In terms of throughput and analyte numbers, Xia notes that bead-based immunoassays have an advantage over plate-based immunoassays. “Bead-based immunoassays are able to detect more targets than surface-based immunoassays as they are not limited by surface size,” he says. “However, while plate-based assays cannot accommodate as many analytes, they are often cheaper and easier to configure, making them well-suited to low plex, smaller scale projects.” The flexibility to customize multiplex targets and optimize the assay for particular experimental conditions are two other factors that weigh heavily in how useful a multiplex platform is for a lab.

Using off-the-shelf products is more common

In general, it is more common for researchers to purchase multiplexed immunoassays from a commercial provider than to develop them in-house. “Developing a multiplexed immunoassay is both complex and time-consuming,” reports Larson. “As well as making sure the antibodies are specific and sensitive, and all the biological components can be mixed together without cross-reacting or causing interference, it is essential to optimize factors such as diluent selection and incubation conditions. Then, you must demonstrate consistent assay performance and ensure others can reproduce your results. By purchasing a multiplexed immunoassay from a trusted supplier, all these variables are taken care of for you, so you can run the assay with ease and have confidence in your data.”

Multiplexed immunoassay data collected from 20 healthy individuals using Bio-Techne’s Human XL Cytokine Luminex Performance Panel Premixed Kit.

“Because most multiplex immunoassays require dedicated instruments, it is usually only specialized researchers who would try to develop these assays themselves,” adds Xia. “These days, hiring a company to develop a multiplex immunoassay for you is relatively cheap. For example, we only charge 30% more to custom print a multiplex assay compared to an off-the-shelf assay, and the per analyte cost is still well below 50% compared to single plex assays.”

xMAP multiplex immunoassays can likewise be customized. “With the Luminex xMAP platform, assay manufacturing partners may have an off-the-shelf product with the required characteristics,” notes Andrada. “If the desired assay is not available commercially, the lab can develop their own; support services and custom assay development labs such as LuminexPLORE Lab can assist with research-based assay development or sample testing services." Importantly, whether selecting an off-the-shelf product or taking a DIY approach, researchers now have more flexibility than ever before when it comes to multiplexed immunodetection.

Tips for developing a multiplexed immunoassay in-house

• Investigate the tradeoffs and caveats for each multiplex immunoassay before deciding which to develop.
• Make sure to fully validate your antibodies in singleplex with a reliable readout, strong model systems, and agreement across orthogonal approaches prior to placing it in multiplex.
• Consider separating targets that do not mix well together across several smaller multiplex immunoassays.
• Think about hiring a biostatistician to handle the data analysis as you proceed higher in plex size.
• Speak with manufacturers for guidance on reagent selection and experimental design.

Provided by Cell Signaling Technology