For the last three decades the predominant means of detecting and measuring antigen-antibody interactions, for research or clinical purposes, has been the ELISA method. While simple, cheap, and relatively specific and sensitive, its hegemony is now threatened by a variety of innovative technologies.
ELISA technology is well-known and widely exploited, depending on an enzyme coupled to a detecting antibody to produce a color reaction in the sample. Despite its simplicity and the lack of expensive reading instruments, it can be tediously slow, requiring multiple wash and incubation steps. Because of this it is virtually impossible to time the steps with accuracy, so the data is quantitative rather than qualitative. Because the plastic surfaces are not homogeneous, proteins will not bind equally to all parts of the same plate, and plate-to-plate variation can be significant. Moreover, contaminating materials in the sample can frequently interfere with the detection system.
Whereas five years ago there was only one label-free detection method available, there are a number of companies exploiting several methods. Label-free biosensors do not require the use of reporter elements, either fluorescent, luminescent, radiometric, or colorimetric, to generate a signal. Their most striking feature is their dissimilarity. A variety of optical and nonoptical methods have been adapted as imaginative researchers have moved to bring new platforms to market.
Surface Plasmon Resonance
Some 15 years ago, Biacore (www.biacore.com) introduced its technology for the label-free measuring of molecular interactions. Based on the principle of SPR, Biacore systems used gold-plated chips coated with a variety of matrices to which proteins or other interaction partners were attached. The total internal reflection of light from the underside of the glass chip was used to measure mass concentration-dependent changes in the refractive index of the bound complex.
At its inception, the first system complemented rather than competed with conventional ELISA detection due to its cost and complexity and was used for determining specificity, association and dissociation constants, affinities, and concentrations of interactants.
Earlier versions of the Biacore system were cumbersome and lacked sensitivity, but over the years new systems have been introduced and performance and ease of operation has substantially improved.
According to Gary Franklin, Ph.D., industrial sector specialist, “I think the ‘Biacore is difficult to use’ myth is a hangover from the early days. In the beginning there were a lot of unknowns in terms of assay design and specific application needs.”
Dr. Franklin added that the early software was an open tool, allowing scientists to examine the potential of unique ways of studying interactions. Consequently, there was less guidance available at that time, so there was a steep learning curve. In close collaboration with the early pioneers, the company gained enormous knowledge and experience, allowing them to develop better and much more user-friendly systems.
“Our Biacore T100 and Biacore A100 systems demonstrate the fruits of these labors,” Dr. Franklin continued. “They provide our highest levels of performance and productivity so far, and customer feedback regarding their ease of use and intuitive performance has been extremely positive.”
Biacore scientists have moved beyond providing SPR as a novel technology to developing a portfolio of systems aimed at a range of key applications. This includes a laundry list of high-quality, label-free interaction analysis systems for the life sciences, pharma/biotech, environmental sciences, and food analysis sectors. These advancements have been achieved through improvements in the user interface and built-in knowledge and guidance of the software.
The two systems, the A100 and the T100, do indeed offer many advantages over Biacore’s earlier models. The T100 is designed for early research through drug development and manufacturing quality control with software for assay development, analysis, and data evaluation and interpretation, whereas the A100 is designed primarily for higher throughput applications and multiplexed analyses, handling up to 3,800 interactions in 24 hours.
The A100 is flexible and can also be adapted for detailed characterization studies enabling integration into multiple phases of project pipelines, Dr. Franklin said. Moreover, the systems are designed for transition into regulated environments with optional packages that provide validation documentation and services.
“These improvements have made the system easier to use,” Dr. Franklin continued, “and this is something we are still striving to develop further as we progress. If you look at what we actually have been offering in the last few years, I think the difficult tag for Biacore is pretty harsh.
“Look at the other high-tech analytical systems, like mass spec and nuclear magnetic resonance. Are these really easy?” he asked. “But then, I suppose initial impressions take a long time to shake off in this business.”
The ForteBio (www.fortebio.com) system takes advantage of the principles of biolayer interferometry. The company’s technology, referred to as Octet, uses biosensor probes through which visible light is passed. The tip of the biosensor is flat and possesses a coated surface that binds various proteins. When the biosensors carrying an attached antigen are inserted in a microplate well they can bind antibody molecules, changing the optical thickness at the tip of the biosensor. This action results in a shift in the wavelength spectrum of light reflected back through the fiber optics of the apparatus, measurable as an nm shift in real time with high sensitivity and reproducibility.
Cantilevers are nanodevices that can be designed to act as sensitive, label-free detection systems. They consist of a gold-coated silicon beam to which antibody molecules (or fragments) can be bound. When exposed to an appropriate solution carrying the antigen, the Ag/Ab complexes will exert a stress on the beam and cause its deflection.
This deflection can be measured by optical methods, by which a laser light source is focused on the cantilever, or electrically, using a piezoelectric sensor in which its electrical resistance changes when it is either compressed or elongated. The mechanical movement of the cantilever is thereby transformed into a detectable electrical signal.
Cantion (www.cantion.com), acquired by Nanonord in 2005, is one of several companies investigating the cantilever technology. Cantion produces the CantiChip8, a sensor element consisting of an array of eight nanomechanical cantilevers. The cantilevers, which are 100-µm long, 50-µm wide, and 0.5-µm thick, are coated on the upper side with a detector layer and are able to recognize and interact with the molecule of interest. The cantilevers have integrated piezo resistors that combine simplicity with sensitivity.
Another company exploring the possibilities of cantilevers is Luna Innovations (www.lunainnovations.com). The company researches, develops, and commercializes molecular technology and sensing solutions. Life sciences director, Rick Obiso, and his group focus on environmental detection and remediation, including bacterial and viral detection.
“Microcantilevers have the potential to form an exquisitely sensitive detection system,” reported Obiso. “Using DNA probes bound to the cantilever, we can detect fewer than one hundred DNA molecules. With an antibacterial antibody, the sensitivity allows detection of as few as a couple of hundred cells.”
Coupling the cantilever technology to the fiber optic sensors offers a number of advantages, since they perform in a fashion, which is not possible with conventional electrical-based sensors. The marriage of cantilevers and fiber optics could result in detector devices that would be handheld, simple to design, and economical.
Label-free Intrinsic Imaging
UV absorption has been a conventional tool for the identification of biomolecules for the last century. DeltaDOT (www.deltadot.com) expanded this detection method into a system it calls label-free intrinsic imaging, a sort of UV detection on steroids. Using extremely small sample volumes (nanoliters), without labeling, it identifies molecules by constructing a space-time correlation as they move across a multipoint detector array. The signal-processing algorithms draw a connection between the position of the biomolecule as it traverses the detector and the time it is detected.
According to Stuart Hassard, Ph.D., co-founder and chief biologist, the company has designed a stable of devices dedicated to different detection tasks. The Peregrine system is a high-performance capillary electrophoresis analyzer that identifies proteins, glycoproteins, peptides, nucleic acids, bacteria, viruses, and small molecules.
The samples are separated and identified through multipoint detection, a technique adopted from high-energy particle physics. Instead of identifying the molecules at a single detection point, as in standard electrophoresis, the biomolecules are detected over and over again as they traverse the detector, which regains the signal-to-noise loss resulting from absence of a label.
A second platform device, the Merlin, performs label-free sequencing of DNA. It is a benchtop instrument that uses DeltaDOT’s signal-processing algorithms to identify standard DNA-sequencing products. The unlabeled DNA extension products are identified using the same space-time correlates as the Peregrine, allowing an increase in throughput by serial multiplexing in a single capillary. The device can be adopted by small laboratories as a benchtop device, similar to a PCR machine, with rapid identification of amplified DNA sequences and considerable financial savings since the use of fluorescent labeling of nucleotides is not required.
Yet another application of the label-free detection intrinsic imaging technology is in the field of forensics and biodefense applications. The platforms can be adapted for detection of pathogens and other chemical and biological weapons.
“We believe the technology lends itself well to applications in portable devices,” Dr. Hassard said, “and the speed and accuracy of the technology offers many possibilities for detection of terrorist threats.”
Akubio (www.akubio.com) uses the piezoelectric properties of a resonating quartz crystal to detect biomolecular interactions. The company introduced its RAP•id 4 instrument at last year’s annual SBS meeting; it is designed for real-time label-free measurement and analysis of biomolecular interactions.
“The physical principle, known as resonant acoustic profiling (RAP), is based on a functionalized resonating quartz crystal, which changes in frequency as molecules bind to receptors,” chief scientist Matthew Cooper, Ph.D., explained.
The instrument contains a microfluidic delivery system, which passes the analyte of interest across the surface. The data generated indicates the presence of the analyte as well as the specificity, affinity, and kinetics of the surface-bound receptor. The measurement of these parameters provides an acoustic profile covering a vast range of molecular interactions.
The instrument measures the build-up of molecules on the crystal surface and is immune to distortions from crude or buffered solutions. It can be applied to a wide range of interactions, including antigen-antibody binding, receptor-ligand pairing, and binding of molecules to bacteria and viruses.
The Akubio technology resolved a number of long-standing problems in acoustical-sensing devices, including poor sample delivery, faulty thermal controls, and the lack of a multisensor analysis option. The current instrument employs microfluidic delivery combined with automated liquid handling.
The Akubio team has modified the surface chemistry in order to reduce the degree of nonspecific binding by effectively masking the chemical and physical properties of the surface with a resulting increase in sensitivity of the system. According to Dr. Cooper, the RAP•id 4 can detect levels of analyte in the ng/mL range with a moderate throughput time frame (400 samples per day).
One of the major trends in label-free detection is a move toward fragment screening and other structure-based approaches that employ significantly smaller compound libraries than traditional high-throughput screening. This development is happening at the same time that the throughput of label-free systems is increasing, which is bound to move label-free more into the screening arena, even for primary screening duties.
Recent advances in label-free detection have great appeal, but there are shortcomings. As with many new approaches, expensive and complex instrumentation is required, which may be beyond the budget of many academic laboratories. Clinical labs would be unlikely to commit to a large expenditure of capital in the absence of a whole panel of tests configured for the particular instrument.
The appeal of conventional ELISA technology is its simplicity and low cost. Reading ELISAs requires a relatively simple instrument, and in the case of lateral flow assays, no instrument at all.
Furthermore, although somewhat long of tooth, ELISA-based technology, when performing optimally, can be simple, inexpensive, and sensitive. Because of its venerability, it will take extensive comparative validation assessing relative sensitivity, cost, repeatability, and convenience to establish the merits of the new label-free detection systems.