December 1, 2014 (Vol. 34, No. 21)

Richard A. A. Stein M.D., Ph.D.

Portable and user-friendly assays can be powerful and
sophisticated, too, detecting transmembrane proteins
and other complex epitopes.

Immunoassays have been at the forefront of biotechnological innovation, driving advances in both the lab and the clinic, sparking scientific insights and guiding diagnostic and therapeutic decisions.

As such, immunoassays represent a technological vanguard, one that possesses the potential, like any vanguard, to mobilize the common run.

To realize this potential, researchers and developers are working to broaden the appeal and applicability of immunoassays. They are improving immunoassay sensitivity and specificity, adding multiplexing capabilities, decreasing costs, and devising platforms that are not only highly capable and readily available, but also portable. These advancements promise to markedly expand the population of end-users benefiting from rapid, inexpensive, and accurate testing. Such testing, extended to resource-constrained settings, may significantly impact global public health.

Essentially, immunoassays are biochemical reactions that qualitatively and quantitatively measure specific analytes from biological fluids. They are among the most frequently performed clinical laboratory tests. Since the 1950s, when the first immunoassays were developed, the dynamic detection and quantitation of antibodies has found increasingly important roles in research and in clinical medicine, for diagnostic and therapeutic applications.

Green-Lighted Liquid-Phase Assay

“Antibody screening is important for many applications, such as tests in infectious and autoimmune diseases, and for personalized and predictive medicine, but at the same time, large-scale arrays that are currently in use can often yield false-negative and false-positive results,” says Peter D. Burbelo, Ph.D., staff scientist at the National Institute of Dental and Craniofacial Research, a branch of the National Institutes of Health.

Dr. Burbelo has developed a new assay class known as luciferase immunoprecipitation systems (LIPS). It relies on a light-emitting recombinant protein to detect antibodies that are present in various medical conditions. “More and more people, from academic institutions to private companies, are becoming interested in the various applications for LIPS,” asserts Dr. Burbelo.

LIPS takes advantage of the luciferase gene, or rather its enzyme, which emits light and is useful as a reporter. Luciferase-based assays are known to have broad linear ranges—they extend over seven orders of magnitude. Proteins, protein fragments, or peptides of interest can be fused to the luciferase reporter gene by recombinant technologies.

“Transmembrane proteins or very complex conformational epitopes are always more challenging irrespective of the technology that is being used,” advises Dr. Burbelo. “Several different flavors, such as deletion mutants or N- and C-terminal fusion proteins, may have to be generated.”

After the fusion protein-encoding plasmids are transfected into mammalian cells, the tag allows crude protein extracts to be used, circumventing the need for protein purification. “Since LIPS is a liquid-phase assay, proteins for the most part maintain their conformation, and one can pick up hard-to-detect antibodies, which typically is done with radioimmunoprecipitation assays that require radioactive materials,” explains Dr. Burbelo.

For performing an immunoassay using LIPS, a specific amount of recombinant protein, quantitated based on light emission, is incubated with the serum of interest. During a second step, the mixtures are transferred to a plate that contains antibody-capturing reagents. After this incubation and several washing steps, coelenterazine, the luciferase substrate, is added to measure light production and determine the amount of antibody that becomes bound to the fusion protein.

“LIPS generates a signal that is often 100–1,000 times higher than the control,” notes Dr. Burbelo. Recently, Dr. Burbelo and colleagues developed a LIPS-based approach to diagnose Lyme disease and revealed that the test provides a 1,000-fold detection span for antibodies, without the need for serum dilution, and with 98% sensitivity and 100% specificity.

The time requirement for a LIPS immunoassay is approximately 2.5 hours, shorter than for ELISA and Western blotting. For several medical conditions, LIPS either provides better diagnostic testing or offers new information when compared to existing approaches.

“We can look at many antigens and many sera at a time, but profiling hundreds of things at one single point at the same time is still difficult with this technology,” admits Dr. Burbelo. “Building LIPS into a more multiplexed or large-scale target discovery is one of the future goals.”

Paper-Based Devices at Point of Care

 “We developed a platform that can easily be used at the point of care, in locations where a sensitive and specific clinically actionable test is needed,” says Charles Mace, Ph.D., assistant professor of chemistry at Tufts University. Dr. Mace and colleagues developed a three-dimensional singleplex paper-based diagnostic immunoassay and illustrated its utility by measuring human chorionic gonadotropin in buffer and urine, revealing that it performed comparably to commercial lateral flow assays.

This project was conducted in collaboration with Diagnostics for All, a nonprofit company that proposes to design and manufacture low-cost diagnostic devices that are easy to use at the point of care and can be specifically useful in the developing world.

“This paper-based application is the first step toward building a platform that enables other immunoassays to be developed for point-of-care testing,” emphasizes Dr. Mace. One advantage of this platform is that it can be used, without extensive optimization, as a building block for a number of different immunological assays.

“Together with our collaborators, we are investigating how agnostic this platform is [with respect] to other kinds of immunological tests, not only singleplex but also multiplex assays,” informs Dr. Mace. “[We intend] to increase the density of information that can be acquired and provide powerful diagnostic information.”

Affinty-Probe-Enabled Nanoparticles

“Nanoparticles, in particular gold colloids, have been used for a long time in assays, and newer products that became more recently available, such as quantum dots, are poised to increase nanoparticle assay sensitivity,” says Matthew A. Cooper, Ph.D., professor of molecular biosciences at the University of Queensland.

Nanoparticle-based materials have found applications in a broad range of diagnostic tests, including immunoassays and nucleic acid assays. However, in complex biological fluids, their performance is often suboptimal due to nonspecific interference from other molecules.

Efforts in Dr. Cooper’s lab are focusing on developing and optimizing nanoparticle-based biosensors, with particular attention devoted to the attachment of affinity probes. This design consideration has implications for stability and functionality.

“Engineering molecules to make nanoparticles stable and functional has been slow,” comments Dr. Cooper. “But we now have a better handle on the chemistry of the attachment process.”

In a recent study, Dr. Cooper and colleagues examined four sandwich immunoassays in which different strategies were used to functionalize the surface of the nanoparticles with affinity probes, and compared them for their ability to detect NS-1, an early biomarker of dengue fever infection. The type of functionalization emerged as an important factor for shaping the performance of the assay.

This experimental approach revealed a 10-fold change in the dynamic range of NS-1 detection between different types of functionalization. In addition to decorating the surface of the nanoparticles with affinity probes, this strategy pointed toward other key determinants of the sensitivity, specificity, and functionality of the assay, such as the choice for the biochemical attachment strategies and the possibility of conferring flexibility to the capture probes.

“One shortcoming is that for newer nanoparticle types, assay development and attachment chemistries are not sufficiently reliable and robust to be  ‘fit for purpose’ in a commercial product,” notes Dr. Cooper. Robust and mature manufacturing processes have been developed for products that have been available for two or three decades, such as magnetic, latex, and gold particles. “Improving the manufacturing process, and making sure that the resultant assays are also robust in real clinical settings, are our main areas of emphasis at this time,” states Dr. Cooper.

Droplet-Based Assays

“We are moving away from the regular microfluidic devices, and more toward using droplets as microfluidics,” says Jeong-Yeol Yoon, Ph.D., associate professor of agricultural and biosystems engineering and biomedical engineering at the University of Arizona. Droplet-based assays provide multiple advantages, including high throughput, small experimental volumes, decreased sample loss, reduced sample contamination, speed, and the possibility of easily manipulating the droplets.

Nevertheless, they also present the inconvenience that hydrophobic surfaces create a slippery environment and increase the vulnerability of the droplets to external vibrations. To establish conditions that would ensure the stability of the droplets under various environmental conditions, including vibrations, Dr. Yoon and colleagues designed a new experimental strategy for droplet-based immunoassays that is powered to detect and quantitate Escherichia coli and Salmonella typhimurium with high sensitivity and specificity.

In this multiplexed fluorescence/light scatter particle immunoassay, droplets are immobilized on a nanofibrous substrate, providing non-slip conditions that ensure that they are immobile and resistant to movement or external vibrations.

The two bacteria can be simultaneously detected within a single-pipetted sample. Anti-E. coli-conjugated green fluorescent particles and anti-S. typhimurium-conjugated red fluorescent particles, which have different excitation and emission wavelengths, allow multiple targets to be detected concomitantly in the same droplet. Using fiber optics-based light sources, Dr. Yoon and colleagues determined a 102 CFU/mL limit for detecting bacterial organisms using this approach.

“In another innovation to this immunoassay, we are one of the first to use the smartphone as an optical biosensor, and this allowed us to make the assay portable,” remarks Dr. Yoon. The assay can detect live or dead bacteria and can be performed under a variety of experimental conditions.

With these characteristics, the assay may be suitable for point-of-care and field testing, and because the assay does not require pretreatment or enrichment steps, it can easily be adjusted for detecting other microbial pathogens. “In the future, we would like to improve the user interface and make this platform easier and more reliable for end-users,” comments Dr. Yoon.

Scientists are improving immunoassay sensitivity and specificity while adding multiplexing capabilities, decreasing costs, and working to devise portable platforms. [Photosketch/Shutterstock]

Epitope-Friendly Cell Screening

“We have developed a high-throughput flow platform that incorporates a flow cytometry engine inside a true high-throughput screening instrument,” says Thomas Duensing, Ph.D., chief technology officer at IntelliCyt. This platform, called iQue Screener, enables investigators to perform assays on cells in suspension, including cells of the immune system.

“The ability to perform high-throughput screening for cells in suspension is a unique capability,” asserts Dr. Duensing. In addition to cells, this platform, called iQue Screener, is able to screen microbes, beads, and mixtures in suspension. “One of the primary applications of our immunoassay platform,” Dr. Duensing adds, “is antibody screening for hybridoma phage-display libraries.”

The iQue Screener can process a 96-well plate in 3 minutes and a 384-well plate in 12 minutes, and a new platform, iQue HD Screener, is able to extend this capacity and process a 1,536-well microplate with cells in suspension within 80 minutes. The platform is optimized for minimal sample usage, and it is able to perform measurements using one-microliter samples.

In antibody screening campaigns, the ability to present antigens on cells and maintain the antigens’ native conformations—that is, preserve epitopes during screening, which is particularly important for certain proteins, such as transmembrane proteins—is one of the factors that shapes the physiological relevance of the measurements. In addition, the ability to analyze a large number of objects per well in a highly multiplexed format contributes to statistical relevance.

“Our platform also enables multiplexed screening assays while offering the possibility of performing several experiments in the same cell and in the same sample to monitor multiple targets,” remarks Dr. Duensing. Consequently, control and target cell lines can be included in every well on a plate. “We can have multiple cell types and multiple cell populations in a well and use markers to differentially identify them and look at the biology of each one of those subpopulations simultaneously,” continues Dr. Duensing.

These internal controls, he maintains, contribute to cost-effectiveness and improve the time sensitivity of many applications: “In hybridoma screening, the source of the library is living cells, and it often is necessary to go back to those cultures and pull out cells that are identified in a screen. There is a time-sensitive nature to that.”

The iQue Screener was critical for findings reported by several groups, including studies on dengue virus immunity, dengue virus drug and vaccine targets, and analyses of the antibody response to malaria infection during pregnancy. “We are constantly improving our platform to make it more user-friendly and capable of analyzing large, high-content datasets,” emphasizes Dr. Duensing.

The software analysis package, because of its flexibility, allows investigators to perform extensive assay development experiments. According to Dr. Duensing, “Investigators can then save the experimental design and analysis they want to use in future screens, set them as a template, and automatically run screening campaigns in a turnkey fashion.”

IntelliCyt’s iQue Screener is a high-throughput instrument that incorporates flow cytometry. The instrument can sample suspended immune cells and other objects, and it can generate multiplexed data—up to six measurements per object. In antibody screening campaigns, iQue Screener can present antigens on cells and maintain the antigens’ native conformations.