September 15, 2016 (Vol. 36, No. 16)
Nothing Beats Immunoassays for Sensitivity, Specificity, and Flexibility, but Users Want More and Vendors Are Delivering
GEN: What makes a long-standing technique like immunoassays so valuable to research laboratories?
Dr. Murray: In short: their sensitivity, versatility, and simplicity. Immunoassays can detect and quantify analytes at picomolar levels in complex biological samples like serum. No other technique is capable of that. They’ve also been developed in several formats to tackle a broad spectrum of questions: single and multiplex assays can be carried out across plate, bead, flow, and other platforms. Finally, many immunoassays can be run on even the most basic lab equipment—just an absorbance microplate reader in the case of ELISAs. This makes a very powerful technique immensely accessible.
Mr. Chan: Immunoassays are essential tests used to detect the presence or absence of large molecules, such as medicines, hormones, or specific proteins. They are used extensively in life science laboratories, either in a R&D or commercial setting, to help identify predictive biomarkers or potential targets for new drug candidates.
Mrs. Yasuda: Immunoassays are valuable because they can accurately identify and quantify new protein analytes for potential biomarkers. Of the various types of immunoassays, ELISA has been proven and tested as the best validated method for measuring individual cytokines. Multiplex assays offer simultaneous detection of multiple analytes in a single reaction thus providing more information on proteins for quick and cost-effective identification of biological processes and the study of disease progression.
Dr. Braunschweig: From the Nobel Prize in 1960 for the first immunoassay measuring insulin levels, to the ELISA in the early 1970s, to the modern development of highly sensitive and specific multiplex panels, immunoassays provide key answers about the roles that proteins and other biomolecules play in a myriad of biological processes and diseases.
Today, the major advantages of immunoassays are their sensitivity, ease of use, flexibility, and low cost. Furthermore, as new proteins become important research targets, the rapid development timeline for new immunoassays accelerates discovery. These factors lead to over 10,000 studies published annually using immunoassays in life science research.
Dr. Wegner: Researchers count on immunoassays to reproducibly measure a wide variety of emerging and established biomarkers. The specificity of antibodies allows for the robust detection of analytes in complex samples such as serum, plasma, cell supernatants, cell lysates, and tissue extracts.
Whether researchers prefer colorimetric, chemiluminescent, or fluorescent detection methods, there are compatible immunoassays available. Multiplexed assays like Luminex and Proteome Profiler™ arrays are optimal screening tools for measuring up to 100 analytes in a single assay, while ELISAs are ideal assays for studies focused on a smaller number of analytes.
Mr. Zock: The value and longevity of proven immunoassay techniques like ELISA is due to the breadth of available antibodies that can be used as reagents enabling an almost endless number of assays to be developed. Antibodies can be used to identify or interfere with active components of proteins, carbohydrates, and DNA including conformation specificity.
Immunoassays are also easy to perform using widely available plate-based peripheral automation like plate readers, washers, and liquid handlers. Adding the commercial availability of reagents and access to expertise, immunoassays continue to be a cost-effective strategy for investigating both routine and emerging biologies.
Mr. Welch: Immunoassays are a critical part of biological research allowing scientists to answer the fundamental questions: is my protein or other macromolecule of interest present in my sample and what is its concentration? Their sensitivity and specificity allows immunoassays to be used in a wide range of applications from basic research to the clinic: providing confirmation of observations made with molecular markers, pharmacokinetic and bioequivalence data during drug discovery, the effectiveness of therapies, and the diagnosis of diseases.
Dr. Mohan: Immunoassays are highly useful in biological research and provide a method to quickly and accurately detect the presence and subcellular location of specific molecules. These techniques rely on the ability of an antibody to bind to the specific structure of a molecule. Immunoassays are also widely used in the diagnosis of diseases, therapeutic drug monitoring, clinical pharmacokinetic, and bioequivalence studies in drug discovery.
Immunoassays are desirable due to their inherent specificity and adaption for sensitive, high-throughput analysis of a wide range of analytes in biological samples. They can also be combined with real-time PCR to detect infectious agents in samples.
Dr. Nath: Immunoassays are probably one of the most widely used analytical methods because of their ability to detect wide range of small molecules, proteins, cells, and other biomolecules with high sensitivity. Immunoassays, especially ELISAs are valuable because they are easy to develop, have the advantage of widely available reagents and instrumentation, are high-throughput, and can be performed in manual or automated format.
Alternate bead-, chip-, and microfluidics-based immunoassay formats have further enhanced the sensitivity, improved dynamic range, and allowed monitoring of real-time binding kinetics.
Dr. Piatti: The study of protein expression is key to many areas of research, such as neuroscience, oncology, development and cell differentiation, and immunoassays still represent the assay of choice. They allow researchers to answer a variety of questions about their target proteins. Depending on the particular immunoassay, you can get apparent molecular weight, assess purity, and quantify expression (relative and absolute).
You can even study post-translational modifications with certain technologies, and multiplexing makes screening for multiple targets a much simpler task. Researchers have so many formats to choose from. Most look for sensitivity, quantitation, and ease of use.
Dr. Jones: The value of immunoassays lies in the high sensitivity and specificity afforded by the detection system, the high-throughput capabilities, and their ability to quantitatively detect a wide range of analytes in a variety of biological samples, particularly those that are complex and heterogeneous.
The ability of immunoassay methods to quantify a wide variety of molecules such as proteins, steroids, drugs, macromolecular biomolecules, metabolites, and biomarkers, which indicate disease diagnosis or prognosis, make them increasingly clinically relevant. The minimal sample preparation and analysis time also make immunoassays well suited for use in research laboratories.
GEN: What is a major obstacle currently facing immunoassay users and how is your company addressing the issue?
Dr. Murray: We know that users need reliable tools in a sector that increasingly demands greater sensitivity and higher throughputs. That’s why our developments work to streamline standard protocols and eliminate sources of error. This helps scientists generate results faster, and more consistently.
RabMAb® and recombinant antibodies at Abcam—currently being knockout validated for specificity— ensure sensitivity and reproducibility. They also form the core of three immunoassay platforms: matched antibody pair kits, SimpleStep ELISA®, and Firefly® particle technology for multiplex assays. Together these provide researchers with the flexibility to efficiently scale their research, without compromising the precision or accuracy of their data.
Mr. Chan: Typically, you need really strong binding and high specificity for immunoassays to work very well, especially in situations where you are using them for diagnostics or for biomarker testing. At Abpro, we’ve been focusing on this aspect for a while to overcome this hurdle by being able to develop antibodies that can be used in immunoassays for applications in diagnostics and therapeutics with these types of characteristics.
We have optimized our automated Divers ImmuneT discovery platform to create antibodies with these kinds of features and our commercial partners have been using our antibodies very effectively now in a variety of immunoassays.
Mrs. Yasuda: With the continuous discovery of new biomarkers, especially in the growing field of immuno-oncology, researchers require the flexibility to analyze new analytes as they become available for their study. ProcartaPlex® Multiplex Immunoassays from Affymetrix address this critical need by offering customization of multiplex panels specifically tailored to the researcher’s study.
ProcartaPlex® Immuno-Oncology Checkpoint Panel, one of the newest panels, can simultaneously quantify 14 key immuno-oncology checkpoint molecules. Furthermore, ProcartaPlex assays and Platinum ELISA from Affymetrix have 98% correlation of antibody pairs demonstrating high correlation, providing the freedom to move from a multiplex assay to an ELISA assay as needed.
Dr. Braunschweig: As biomedical research progresses, scientists are looking for new immunoassays that allow more efficient sample usage while maintaining sensitivity and accuracy. Using traditional immunoassay techniques, researchers frequently must choose only a few biomarkers to measure, thus limiting their ability to investigate the complex interaction between many biomolecules.
Bio-Rad Laboratories provides a solution to this obstacle with the Bio-Plex portfolio of multiplex immunoassays using Luminex xMAP technology. With Bio-Plex, researchers can perform up to 40 immunoassays in a single well, while maintaining the sensitivity and accuracy of the traditional ELISA, from sample to answer in about four hours.
Dr. Wegner: Researchers are under pressure to deliver results quickly, but they still need reproducible sensitivity. The Ella platform from Protein Simple, a Bio-Techne brand, fully automates single or multi-analyte ELISAs. Simple Plex™ assay cartridges provide results in less than an hour with pg/mL sensitivity and a 4-5 log dynamic range.
The multi-analyte cartridge is compatible with four immunoassays. Samples are split across microfluidic channels completely eliminating any potential cross-reactivity. Only 25 µL of sample is required and since there are no manual wash-steps there’s almost no biohazardous waste.
Mr. Zock: One major obstacle is the lack of multiplexing options for immunoassay formats like standard ELISA. This limits the amount of information “content” that can be extracted from a given biological sample. Our company overcomes these limitations by providing multiplexed single cell and bead measurements. We retain the ease of use of plate-based formats for screening large sample numbers but with dramatically reduced reagent and sample usage to make it more cost effective.
By measuring cells and beads together immuno-phenotyping, cell function, and secreted protein profiling can routinely be done in the same wells.
Mr. Welch: An obstacle facing immunoassay users is the ability to reliably and reproducibly measure combinations of proteins expressed at low levels in complex sample matrices. This requires assays that are sensitive, can specifically measure the analyte of interest, provide the same result each time the assay is used, and can be combined in a multiplex format.
Building on the strength of our electrochemiluminescent technology, which provides exceptional sensitivity, we have developed our V-PLEX multiplex assays under design control and according to the FDA’s analytical validation guidelines. Combined with rigorous manufacturing and QC specifications this ensures sensitive, specific, reproducible results.
Dr. Mohan: Despite the analytical sensitivity of immunoassay, a few challenges still exist that affect performance of antibodies. One of the major contributing factors to the variability in immunoassays is the difference in antibody development, purification, and validation techniques used by different vendors.
Antibody specificity can be affected by interference caused by polyreactive antibodies and heterophile antibodies that may interfere in a noncompetitive manner by binding to conjugates, enzymes, or other components of the detection system. At MilliporeSigma, we have established tight controls and extensive multiple validation protocols using positive and negative controls to qualify antibodies for various immunoassays.
Dr. Nath: Key obstacles facing immunoassays are (A) narrow dynamic range that limits their use especially in pharmacokinetics studies of therapeutic antibodies, (B) heterogeneous format involving multiple wash and incubation steps, which limits the throughput and require significant time investment, and (C) most immunoassays use protein fragments for antibody screening resulting in selection of antibodies that often fail in downstream biology.
Recently, Promega developed an ultra-bright bioluminescent protein called NanoLuc that promises to bring significant improvements to the immunoassays. NanoLuc can be recombinantly or chemically conjugated to the antibodies, which results in immunoassays with wide dynamic range and high sensitivity.
Moreover, NanoLuc enables a cell-based Bioluminescence Resonance Energy Transfer (BRET) immunoassay which is homogeneous (no wash assay) and utilizes cells with fully folded membrane proteins. This format is suitable for high-throughput antibody screening in the right biological context.
Dr. Piatti: Major hurdles are time to results, reproducibility, and sample size. Simple Western™ is the only gel-free, blot-free, hands-free capillary-based immunoassay platform that integrates and automates the entire protein separation and detection process. It separates and analyzes proteins by size (from 2 to 440 kDa) without a blotting step so inconsistencies caused by protein transfer are eliminated. With Wes™ you only need 5 µL of starting material to analyze up to 25 samples in 3 hours flat. And there’s a charge-based assay if you’re doing post-translational modification characterization. Our software automatically analyzes assay data, and offers 21 CFR Part 11 compliance tools.
Dr. Jones: One of the greatest obstacles currently facing immunoassay users is the lack of necessary sensitivity to analytes at levels reaching biological significance. Many proteins important in cancer, neurological disorders, early stages of infection, etc. are present at levels below standard immunoassay levels of detection.
Our company is using a new technology, single-molecule enzyme-linked immunosorbent assay, to isolate and detect single protein molecules in complex biological samples allowing for measurement of extremely low (sub-femtogram) concentrations. This 100 to 1,000-fold increase in sensitivity coupled with our over 2,600 target pool will allow for increased detection and understanding of numerous diseases.
Immunoassays and Mass Spec
Researchers and clinicians alike are now turning to mass spectrometry as an alternative analytical technique over ELISA-based methods because of its inherent specificity advantage as well as multiplexing capabilities, according to Michael Pisano, Ph.D., vp of biochemistry at Cayman Chemical.
To explore the limitations and benefits of each assay format, he reports that a comparative study of small molecule, competitive ELISAs to that of targeted LC-MS/MS was performed and the results presented in a technical brief (www.caymanchem.com/Literature/applicationnotes/800147).
Seven different primary eicosanoids from either conditioned cell culture media or tissue homogenate samples were quantified with seven individual analyte-specific ELISA kits, as well as with multiplexed LC-MS/MS methods. Measurements from conditioned media matched closely between ELISA and LC-MS/MS, though in this preliminary study, some of the analytes were detected by LC-MS/MS at levels below their limits of quantitation (LOQ), compelling the need for extremely sensitive mass spectrometry equipment when measuring endogenous markers, pointed out Dr. Pisano.
Experimental Data
Data from mouse liver and lung samples were more variable with only four out of seven analytes being comparable between methods. As eicosanoids are particularly challenging to quantify, the significant variability observed between methods from more complex matrices merits further understanding, he continued.
“The inherent specificity and multiplex capability of MS is often thought to be advantageous over quantification techniques that are reliant on antibodies with potentially variable performance characteristics,” Dr. Pisano added.
However, the authors of the technical paper cite the value of antibody-based techniques, acknowledging their importance as partitioning tools to allow detection of less abundant analytes and isolation of structurally related molecules. To improve the sensitivity of MS-based assays, antibodies are proposed for use as an analyte enrichment tool prior to LC-MS/MS in a technique known as immunoaffinity mass spectrometry.
“Thus, both techniques can be quite valuable research tools—one enhancing the limitations of the other—providing specificity and accuracy in analyzing biomarkers in various biological samples,” said Dr. Pisano.
Immunoassays for Traumatic Brain Injury
Traumatic brain injury (TBI) is a leading cause of death and disability in people under age 45. The absence of validated biomarkers in the TBI field is a major factor limiting our understanding of the natural history and long-term effects of TBI, as well as a major barrier to drug development in this area.
Glial fibrillary acid protein (GFAP), ubiquitin c-terminal hydrolase L1 (UCH-L1), as well as other proteins including microtubule-associated protein tau, and amyloid beta peptide have been proposed as promising diagnostic and prognostic biomarkers in TBI. Until now, current assays have not been sensitive enough to reliably detect levels of several key neurological biomarkers in plasma, according to David Hanlon, Ph.D., director of strategic marketing & collaborations at Quanterix.
Utilizing single-molecule detection technology, sensitive immunoassays were developed in Quanterix’ Accelerator Lab for both GFAP and UCH-L1 with an achievement of sub-pg/mL sensitivity, said Dr. Hanlon. Both assays were fully characterized and validated for performance, including lower limits of quantitation (LOQ), linear range of calibration, spike recovery, and dilutional linearity.
Using each of the optimized assays, a total of 90 clinical samples from three different diagnostic groups (normal, mild-moderate TBI, and severe TBI) were analyzed for GFAP and UCH-L1, respectively.
“While these evaluated samples showed both proteins to be useful potential TBI biomarkers, GFAP exhibited a better statistically significant separation than UCH-L1 between normal and TBI samples,” he continued.
Further, in a separate study (J. Neurotrauma, in press), researchers demonstrated that in samples collected shortly after a suspected concussion (day 0), as well as at days 30 and 90, GFAP was significantly elevated soon after injury (day 0) and was an excellent biomarker for the discrimination of complicated mild TBI (mTBI) from controls.
“The development of these highly sensitive immunoassays for GFAP and UCH-L1 has been critical in advancing early detection of TBI and mTBI to avoid neurodegenerative disease associated with multiple head injuries, such as chronic traumatic encephalopathy,” said Dr. Hanlon.
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