September 15, 2018 (Vol. 38, No. 16)

Taking in “Everything All at Once” Can Reveal Meaningful Biological Patterns and Drug Targets

Multiplexed immunoassays invite us to unleash our inner nerd and, in the words of Bill Nye the Science Guy, examine “everything all at once.” A nerdy obsession with details, Nye insists, can help fill out the big picture—provided the details can be seen in the same frame without too much loss of resolution. For example, the details called analytes—or rather, analyte signals—can be used to distinguish between disease states and healthy states, provided they are not allowed to interfere with each other or blur into the background.

Managing this balancing act—tracking multiple analytes while keeping them distinct—is the essence of the multiplexed immunoassay, a technology that uses antibodies to detect and quantify proteins and other kinds of molecules in complex samples. Usually, the antibodies coat elements such as beads or microwells. Properly configured, such elements, along with reagents and detector technologies, can allow dozens of analytes to be quantified in a single assay, saving investigators the trouble of running many singleplex assays—and saving precious sample volumes, too.

Streamlined workflows, such as the ones described in this roundup article, can expedite the characterization of disease states, the development of biomarkers, and the screening of drug candidates. Besides offering efficiency, multiplexed immunoassays can help investigators glimpse otherwise hard-to-see biology. For example, they can reveal complex interactions within or between biological pathways, sharpening diagnostic procedures and advancing the development of powerful therapeutics.

Multiplexed immunoassays, then, may achieve what Bill Nye had in mind when he wrote, “Try to take in all the details—everything all at once—and then sift through them to find the meaningful patterns, as part of an effort to make the world a little bit better.”

GEN: What are the main advantages of multiplexed immunoassays over other assays?

Dr. Murray: In contrast to singleplex immunoassays that measure only one analyte per assay, multiplex immunoassays can provide more in-depth data by measuring many analytes at once, often in less time and from less sample than with singleplex systems. Another attractive feature is that multiplex assays continue to be increasingly miniaturized, with 96-, 384-, and even 1536-well plates that use just microliters of sample, without changing plate size. That’s a lot more data from less sample.

Dr. Balderas: Multiplexed assays add a variety of advantages for the scientist today. These advantages range from the ability to obtain a large amount of data from a small sample (blood, urine, tears, synovial fluid, etc.). A second advantage is ability to perform complex assays with multiple analytes in a single test as compared to the need to perform individual tests…thus providing less user error. Additional cost savings in workflow and reduced user time are also advantages of multiplex assays.

Dr. Jordan Dreskin: One of the main advantages of multiplexed immunoassays such as Bio-Plex Pro immunoassays over traditional ELISAs is the ability to detect many targets from a very small sample volume in a single well. These assays are also developed and optimized using a rigorous design process using biological samples to ensure that analytes are accurately detected across a broad assay working range. This allows you to detect both low-level endogenous and high-concentration analytes in a single experiment, saving you time, sample, and hands-on steps.

James Murray, Ph.D. Director of Immunoassay Development, Abcam

Overall, choosing an optimized multiplex assay allows you to focus on getting results rather than going through the assay performance optimization yourself, saving you time while obtaining sensitive, accurate results.

Dr. Mao: Multiplexed immunoassays offer several advantages. For example, multiplexed immunoassays can:

1. Save time: High-throughput multiplexed immunoassays enable researchers to gather more information in less time.

2. Reduce costs: A multiplexed assay is more affordable per analyte-sample pair than singleplex ELISA (that is, >$1 versus >$10, respectively).

3. Reveal the “big picture”: Multiplexed detection can profile biomarkers that exist in a network and work in synergy with each other.

4. Require less sample: Less biological sample is needed to analyze hundreds and even thousands of analytes at a time.

Dr. Sebata: Immunoassays that combine multiple analytes in a single reaction volume, such as the Thermo Fisher Scientific ProcartaPlex assays on the Luminex platform, produce rich biological information from smaller amounts of sample. These assays can multiplex up to 80 analytes in one assay, which is equivalent to running 80 separate ELISAs. Multiplexing assays provide enhanced solutions to both workflow and sample limitation problems by eliminating the need to perform parallel individual measurements. Centralized kit reagents and reduced sample preparation minimize immunoassay variability as well as user/experimental errors.

Yingqing Mao, Ph.D.
Senior Director, R&D, RayBiotech

GEN: What are some specific applications ideally suited for multiplexed immunoassays?

Dr. Murray: Multiplex immunoassays do two things exceptionally well: (1) accurately quantifying low-abundance proteins in very complex biofluid samples, and (2) using panels to measure a host of proteins simultaneously. This resolution and flexibility make multiplexing particularly well suited to immunology, cancer, immuno-oncology, and biomarker discovery where a series of different proteins (a panel) might be measured together as an indicator for a specific disease, or as a response to medical treatment.

Being able to look at multiple cytokines or other signaling molecules at once, as well as internal controls, means researchers can investigate signaling pathways with greater clarity. While single-target investigations of the past might have thrown up anecdotal or isolated data from one analyte, multiplex investigations can reveal complex interactions within or between pathways—for immune responses or discovery processes, for example.

Dr. Balderas: One of the most common applications is the use of bead-based assays such as the Luminex and BD Cytometric Bead Array applications for measuring soluble proteins in complex biological samples. These include proteins such as cytokines, chemokines, and intracellular proteins.

Other applications include plate-based assays that incorporate electrochemiluminescence technology (such as Meso Scale Discovery’s Multi-Array and Multi-Spot assays) or chemiluminescence technology (such as Bio-Techne’s Proteome Profiler antibody arrays). With such technologies, multiple protein analytes can be assessed during drug development, biomarker discovery, and patient stratification efforts.

Dr. Jordan Dreskin: Optimized immunoassays are extremely beneficial in studies where sample volumes are limited, such as mouse studies, tumor biopsies, and neonatal studies. Any study where detection of multiple analytes is a goal will benefit from a multiplexed immunoassay in terms of savings of sample and time, and the benefits are exponential as the study size increases.

Multiplexed immunoassays are incredibly valuable discovery tools that you can use to screen for analytes involved in complex pathways. Bio-Plex Pro Multiplex Immunoassays are developed using a data science process to incorporate analytes within related biological networks, allowing you to cast a broad net to make interesting discoveries at a pathway and system level.

Dr. Mao: Sandwich-based antibody arrays are commonly used in biomedical research to profile analytes representing specific physiological and pharmacological processes. Lower-density arrays (that is, below 40-plex) have proven utility in diverse research areas including cancer biomarkers, autoimmune diseases, neurodegenerative processes, metabolic and signaling pathways, allergies, and HLA antibody screening.

Additionally, pharmaceutical companies use the technology for polyvalent-vaccine preclinical and clinical trials, as well for clinical diagnostics. With increasing affordability of multiplex detection, medium-density (40- to 400-plex) and high-density arrays (>400-plex) are becoming increasingly popular for biomarker profiling and drug discovery.

Dr. Sebata: Given their sensitivity and versatility, multiplex immunoassays are suitable for screening applications in biomarker/drug discovery, as well as in immune monitoring. For example, biomarker discovery workflows frequently utilize donor blood and tissue samples that may be difficult to obtain in sufficient volumes. Multiplexed immunoassays enable the screening of multiple analytes to streamline the discovery process of predictive biomarkers.

Another example includes profiling drug candidate effects on the biological function of the immune system. Detecting small changes in critical soluble protein levels can provide valuable insights into the efficacy of drugs (or drug targets) to modulate important immune system checkpoints.

Robert Balderas, Ph.D.
Vice President of Market Development, BD Biosciences

GEN: What challenges must be addressed if multiplexed immunoassays are to be used efficiently and successfully?

Dr. Murray: One of the biggest challenges for multiplex immunoassays is overcoming the unpredictability of protein chemistry in an assay’s components. Protein chemistry is highly variable, and as the “plex size” (number of analytes being measured) increases, the total number of proteins in the mix also increases, along with the potential for unwanted protein–protein interactions.

A 40-plex, for example, equates to 40 analytes, 40 capture antibodies, and 40 detector antibodies. Consequently, the cumulative potential of nonspecific binding can be much higher, which could mean higher background signal and unwanted off-target interactions.

Abcam’s FirePlex® technology solves this issue by using preoptimized recombinant antibody pairs. Being monoclonal, the antibodies exhibit minimal cross-reactivity and are sensitive and specific to their target analyte, ensuring low variability in protein measurements between runs as well as high batch reproducibility.

Dr. Balderas: The challenges posed by multiplexed immunoassays can be addressed in several ways:

1. Establishing the quality, specificity, and cross-reactivity of the antibodies in antibody arrays to provide unique measurements for each analyte.

2. Determining the optimal number of analytes that can be measured in a single test to ensure that protein–protein interactions not measured in singleplex assays do not occur.

3. Balancing assays across different dynamic ranges for each individual assay in a complex mixture.

4. Establishing a common assay workflow for the kinetics of all assays in the multiplex system.

5. Building efficient protein standards for all analytes in the multiplex system.

Dr. Jordan Dreskin: Choosing the right assay is the first step to success. Understanding how an assay is designed and developed gives you the power to pick the best-fit assay for your application.

To be successful, an assay should meet your needs in terms of sensitivity, be optimized to minimize background and cross-reactivity, and be designed to eliminate sample interference and to detect even subtle differences between diseased and healthy controls. These assays deliver actionable results that can accelerate your research.

Outside of assay selection and independent of assay platform, careful and consistent sample collection, processing, and storage methods should be included in the experimental design. Sample quality is critical to any experiment, so you should be mindful of your sample type and the lability of your target analytes regardless of which assay platform you choose.

Dr. Mao: Carrying out multiplex immunoassays means attending to the following tasks:

1. Choose the right type of multiplexing: Sandwich-based assays generally have higher detection sensitivity and specificity than competition or label-based assays using only one antibody.

Elizabeth Jordan Dreskin, Ph.D., R&D Manager, Applications, Bio-Rad Laboratories

2. Obtain the optimal balance between throughput and assay quality: Increased multiplexing in a single panel generally increases the risk of nonspecific cross-reactivity and decreases assay sensitivity. Antibody colocalization technology can assist in constructing a high-density, highly specific multiplex assay capable of detecting hundreds of analytes at a time.

3. Maintain assay precision and accuracy: Optimize assay diluents to minimize possible matrix effects. Proper sample dilution is also an important consideration.

Dr. Sebata: Challenges with multiplexing immunoassays include (1) a steep learning curve due to more technical handling, (2) more complex instrumentation and analysis, and (3) more difficulty in interpreting the results. Technical and field specialists are often available to teach best practices and provide “tips and tricks.” Instruments should always be calibrated and properly maintained. In-house controls and experimental designs should be carefully considered to aid in the interpretation of the results.

Thao Sebata, Ph.D.
Senior Product Manager, Immunoassays, Life Science Solutions, Thermo Fisher Scientific

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