Choosing the Right Immunoassay for Your Research

There are multiple factors to consider when selecting an immunoassay that best meets the needs of your research. This article reviews those factors and also looks at some of the different immunoassay formats available and shares published examples of their use.

Key factors for immunoassay selection

“In my experience, there are several key factors to evaluate when deciding on a suitable immunoassay format,” reports Paul Wylie, Director, Multiplex, at Abcam. “These include the speed, efficiency, and accuracy requirements of the workflow, as well as how many samples and target analytes are to be investigated. Researchers should also think about the necessary sensitivity within the chosen sample type, as different immunoassay formats vary significantly in terms of sensitivity and dynamic range.” Other important considerations include the cellular localization of the target, the available sample volume, and the ability of the immunoassay to deliver consistent results.

Factors to consider when selecting an immunoassay format

• Workflow speed and efficiency/hands-on time
• Biological relevance and reference ranges—is the immunoassay validated for your target analyte and sample type?
• Number of samples—is an automatable approach required?
• Number of target analytes—is multiplexing necessary/possible?
• Required sensitivity and specificity
• Available sample volume
• Readout—quantitative, semi-quantitative, or qualitative?
• Dynamic range—will samples need to be diluted?
• Cellular localization of the target
• Assay performance and reproducibility
• Available in-house platforms and expertise
• Cost

Multiplexing and discovery research

Deciding whether to go down the multiplex route can be a major discussion point for project teams. Megan Larson, Senior Product Manager, Immunoassays, at Bio-Techne, notes that multiplexed immunoassays are often a preferred choice for discovery research, where platforms such as R&D Systems’ Proteome Profiler™ Antibody Arrays and Luminex’ xMAP assays enable measurement of tens or even hundreds of analytes per sample. “Biology is inherently complicated, with numerous proteins and pathways being closely intertwined, and there is often heterogeneity between individuals or even within the same individual,” she explains. “Multiplexing helps researchers narrow down which targets to pursue and can ultimately save time, money, and precious sample material.” Within the realm of drug discovery, multiplexing provides similar benefits by halting the progression of any compounds that are likely to fail.

Applications of multiplexing

• Using Luminex’ xMAP technology to measure 47 serum proteins in samples from 107 non-small cell lung cancer (NSCLC) patients, researchers have identified a diagnostic panel of 15 biomarkers for lymph node metastases

• During development of an improved method for large-scale production of iPSC-derived macrophages for drug screening applications, researchers relied on Abcam’s FirePlex^® technology to measure 10 different cytokines for cellular characterization

• Using Bio-Techne’s Protein Profiler™ Antibody Arrays to measure relative protein expression levels, researchers have discovered dominant signaling cascades and apoptotic mediators for cadmium-induced cytotoxicity in human bronchial epithelial cells

• By combining Luminex’ xMAP technology with follow-up screening using Bio-Techne’s Simple Plex, researchers have established key similarities and differences between the “cytokine storms” seen in acute dengue fever and COVID-19

Single analyte assays remain popular

While multiplexing offers many advantages, single analyte assays remain hugely popular. “I believe there are two main reasons for the institutionalization of single-analyte assays such as ELISA and western blot,” comments Heather Darby, Scientific and Applications Marketing Manager at Luminex. “First, they are cheap. Second, they have been considered the gold standard of proteomic testing for decades. However, almost any single protein assay can be migrated to the xMAP platform, where switching—even for a single analyte—can provide significant savings in reagent usage compared to these other techniques. Although workflow and sample requirement are similar for single-analyte assays between xMAP and ELISA, xMAP soundly outperforms western blot in both these areas.”

Over time, established technologies for single-analyte analysis have evolved to become faster and easier to use. For example, improved versions of ELISA include off-the-shelf kits that provide results in just 90 minutes with only one wash step (e.g., Abcam’s SimpleStep ELISA^® range) and fully automated ELISA workflows (e.g., Bio-Techne’s Simple Plex platform). Laurence Loï, Product Manager, Immunoassay, at Abcam, reports that a defining feature of SimpleStep ELISA^® is the types of antibody pairs it employs. “Sandwich ELISA is the assay of choice for single-analyte measurement due to its high specificity and sensitivity,” she says. “By manufacturing fit-for-purpose antibody pairs and making these available across a range of different immunoassay formats—including SimpleStep—we enable researchers to move easily from a singleplex, low-throughput assay to a multiplex, high-throughput version, and vice versa, as required.”

Assay chemistry is critically important

According to Dr. Tobias Polifke, Co-Founder and Managing Director at CANDOR Bioscience, the chosen assay chemistry can often have a far greater impact than the choice of assay format or device. To put this into context, he describes a recent customer project aimed at identifying a biomarker for a particular type of cancer. “By initially comparing protein expression profiles of both normal and diseased samples, we were quickly able to home in on a potential target,” he says. “But, when we developed an ELISA for its detection, we could only achieve acceptable coefficients of variation when we diluted the sample material in LowCross-Buffer^®—a reagent designed to minimize nonspecific binding, cross-reactivities, and matrix effects. We subsequently found that, in other buffers, the protein formed various complexes that prevented its antibody-based detection.”

He adds that further studies have shown neither the first protein of interest nor one of its binding partners to be a reliable biomarker for the cancer in question. Instead, only a correlation of the absolute concentrations of both binding partners in relation to one another (after disruption of any protein complexes) provides accurate results. “Unwanted effects such as masking, non-specific binding, and matrix effects can only be addressed through using the right assay chemistry,” Polifke says. “Yet, diluents such as LowCross-Buffer^® are readily applied on any platform, regardless of whether it is a traditional, single-analyte ELISA or a more elaborate multiplexing system.”

Tips for antibody selection

Antibodies form the core of any immunoassay and, when developing an assay in-house rather than using a preconfigured platform, there are certain factors to bear in mind for antibody selection. Loï recommends checking that antibodies are validated for your chosen species and application, stressing the importance of using matched antibody pairs where relevant, while Larson suggests that researchers always perform validation studies in their own model system. “Testing different assay buffers and measuring linearity and recovery will help identify whether you have a specific assay,” she says. “It’s also essential to carry out inter- and intra-assay precision testing to ensure that when the assay is performed by another user, the same results are achieved.”

Polifke notes that as well as minimizing unwanted background signal, LowCross-Buffer^® can provide a form of quality control. “Only high affinity antibodies will bind their intended target in both LowCross-Buffer^® and PBS-T,” he says. “Comparing antibody performance in both buffer types therefore allows you to eliminate low affinity antibodies that may cause problems downstream.” Lastly, Darby mentions considering the purpose of the assay when it comes to antibody selection. “Choosing antibodies that are in a format suitable for their intended use, such as pre-labeled with fluorophores and in compatible buffers, can save a lot of time,” she says. “Additionally, if the assay will be commercialized, the antibodies must be licensable and available in consistent, long-term supply.”

Considerations for antibody selection

• Identify antibodies that are validated for your chosen species and application
• For pair-based assays, select antibodies that recognize different epitopes
• Always perform validation and optimization in your own model system
• Consider using LowCross-Buffer^® for identifying high affinity antibodies
• Select antibodies in a format compatible with their intended use
• Consider recombinant antibodies for guaranteed long-term availability and scalable supply
• Choose licensable antibodies where the immunoassay will be commercialized