BB&T Contributing Editor

SAN JOSE, California – The 38th annual Oak Ridge Conference, sponsored by the American Association for Clinical Chemistry (Washington), held here in April, is a leading forum for presentation of emerging technologies in clinical diagnostics, and has historically provided a window on the technologies shaping the future of diagnostic testing. As discussed by Michael Pringle, PhD, chair of the conference’s opening session, a recent survey found that 56% of the technologies that have been presented at past Oak Ridge conferences were brought to market in a three- to five-year time frame.

At this year’s gathering, highlighted areas included new immunoassay technologies, advanced sensor technologies, and technologies with applications in cancer diagnostics. New immunoassay formats that avoid the need for labels are under development which promise to enhance detection capability and simplify testing processes. Microarray and lab-on-a-chip technologies, including miniaturized devices employing microfluidics, were also described at the conference, along with new molecular diagnostic technologies that are expected to play a role in one of the most rapidly expanding segments of the clinical diagnostics market.

Single-molecule detection, which could have applications in cancer diagnostics by allowing detection of rare events in mixed populations, and could also help simplify sample processing, was another new technology described at the conference and is moving closer to being applied in the clinical laboratory. Many of the newest innovations in diagnostics are being developed not by established leaders in the field, but rather by emerging companies with a strong technology focus. As a result, acquisitions and partnerships will continue to play a key role in the commercialization of the next generation of diagnostic technology.

Immunoassay technology debuts

A number of new developments in immunoassay technology were described at the Oak Ridge Conference. The market for immunoassay products continues to expand, as shown in Table 1, and now comprises the largest segment of the clinical diagnostics market worldwide. The market is increasingly dominated by chemiluminescence immunoassay technologies, with all the leading suppliers in the segment now having a position in that technology. However, emerging approaches now under development are in most cases employing other types of labels for immunoassay detection, or in some cases eliminating the use of labels completely.

For example, Quadraspec (West Lafayette, Indiana), a 25-person spin-off from Purdue University (also West Lafayette), is developing a new immunoassay system based on its BioCD technology, which employs optical interferometry to detect binding of target analytes to the surface of an antibody-coated compact disc. Antibodies specific for the target are spot-deposited on the disc in microwells to serve as recognition elements, and read-out is performed either in a static or flow-through mode, employing signal analysis techniques to extract analyte-specific binding data. The latter read-out mode allows high-speed multiplex assays to be performed, and also results in higher detection sensitivity, according to Joerg Schreiber, PhD, of Quadraspec, who discussed the technology at the conference.

At present, assay sensitivity of 1 nanogram per ml has been achieved in static mode in a model IgG assay, but Schreiber says that the sensitivity can be improved to the one picogram per ml range by employing flow-through detection. The total read time for the assay is two minutes. Up to 80 samples can be analyzed on a single disk if a single-analyte assay is performed.

Alternatively, the company is developing a multiplex assay version of the BioCD that will allow at least 30 different assays to be performed on one disk. Quadraspec has built a production line to manufacture the BioCD devices, and will initially target high-volume/high-throughput applications such as reference laboratory testing.

The BioCD is one of a number of label-free detection technologies under development with potential applications in immunoassay as well as in molecular diagnostics. Table 2 on the following page highlights development-stage label-free assay technologies, some of which were described at the conference.

Label-free detection

Innovative Micro Technologies (Santa Barbara, California) is developing a unique sensor platform for label-free detection that employs microcantilever resonators with internal microchannels. As opposed to conventional microcantilever sensors operating in a fluid sample, which are subject to damping and mass entrainment, the microchannel resonators are operated in vacuum, with the sample introduced into the microchannel. Both large and small molecules can be detected on a single platform, and measurements can be multiplexed to allow several assays to be run in parallel. Typically, dual channels are employed to allow background effects to be cancelled out. Sensitivity is superior to that achievable with related sensors such as surface plasmon resonance detectors, according to Ken Babcock, who described the technology.

Applications have been evaluated for detection of Plasmodium falciparum, the causative agent of malaria, anthrax, botulism toxin, various bacteria, and prostate specific antigen. Although optical detection using lasers has been used initially, piezoresistance detection can also be used, providing added ruggedness and reducing system size.

Babcock estimates the cost per device at between $2 and $5, making it feasible to employ the technology in diagnostic applications. Innovative Micro Technologies has been formed to commercialize the technology for diagnostic and analytical applications.

L. Huang of Akubio (Cambridge, UK) described recent developments using the company’s Resonant Acoustic Profiling (RAP) technology for rapid, label-free detection of bacterial pathogens. The RAP sensor employs immobilized antibodies on the surface of an acoustic wave sensor to bind a target of interest. A second antibody can be added which also binds the target and increases sensitivity by 100-fold.

As few as 1,000 bacteria per milliliter can be detected in complex matrices such as serum and urine. Analysis time is less than 20 minutes. Concentris (Basel, Switzerland) is another company involved in developing label-free assay technology for diagnostic assays. As discussed by Martin Bammerlin, PhD, of Concentris, the company’s microcantilever technology provides label-free detection for immunoassays and can also be used for analysis of DNA and bacteria.

The response to binding of a target molecule results both from mass changes as well as changes in conformation of the receptors attached to the sensor surface. The Concentris Cantisens arrays have also been used to detect the growth of bacteria by detecting the change of mass as the Concentris organisms multiply and accumulate water. A rapid antibiotic susceptibility assay has been demonstrated that can detect growth within two hours.

Particles and surfaces

James Prober, PhD, of DuPont (Wilmington, Delaware) described a new particle-based diagnostic platform for label-free multiplexed assays with potential applications in clinical diagnostics. The Free Array system, described publicly for the first time at the Oak Ridge Conference, employs 20 micron-40 micron particles as optical sensors for detection of binding of molecular targets. Analyte-specific surface capture probes are attached to the particles.

When analyte binding occurs, the refractive index near the particle surface is modified. That change is detected optically by illuminating the particles with a diode laser over a 10 nanometer wavelength range, and detecting the scattered light using a CCD camera chip.

Detection is based on the change in resonance wavelength of the particle. A microfluidic assay system has been developed that allows the particles to be exposed to the analyte solution and assembled within a lattice in a detection cell. A low-volume microfluidic IgG assay has been prototyped having a sensitivity of 500 picomolar and a turnaround time of less than 30 minutes.

Dominique Gorse, PhD, of Bio-Layer (Eight Mile Plains, Australia), described a surface modification technology that produces protein-selective binding without employing antibodies. The surface coatings are formed from multiple components. By themselves, the individual components do not provide high avidity binding of targets, but in combination avidity equivalent to that of an antibody is achieved.

The advantage of the Bio-Layer technology is high stability since the target-binding layer is entirely synthetic, as well as improved performance under harsh conditions, making the technology attractive for testing outside of the central laboratory.

New labeling strategies

New immunoassay technologies employing various labeling techniques were also discussed at the conference.

New nanoparticle labels for use in time-resolved fluorescence assays were described by BioPhysics Assay Laboratory (BioPAL, Worcester, Massachusetts). The company’s R-dots are 45 nm in diameter, while T-dots are 20 nm in diameter. Because of their small size, large numbers of particles can be attached to a single antibody or hapten molecule, enhancing sensitivity relative to conventional labels which typically allow less than 10 reporter molecules to be linked.

In addition, the particles incorporate time-resolved fluorescent labels, allowing increased sensitivity since background effects are reduced. The ability to detect as few as 10 R-dots per milliliter has been demonstrated.

Another new nanoparticle-based immunoassay technology has been developed by Nanosphere (Northbrook, Illinois), the Biobarcode assay. Nanosphere has developed a fully automated platform for ultrasensitive detection of proteins and nucleic acids. The system has been shown to have a sensitivity that is 1000-fold higher than ELISA, and employs magnetic beads for bound/free separation along with labeling of captured protein targets with gold nanoparticles containing tens to thousands of barcode DNA sequences.

The DNA molecules are released in the final step of the assay, providing a high degree of signal amplification. A sensitive detection system is used that leverages the optical properties of gold nanoparticle labels. Applications include high-sensitivity PSA assays, a sensitive assay for the cardiac marker troponin I, and an assay to detect hepatitis B DNA.

Panbio (Brisbane, Australia) has developed a homogeneous assay platform based on Forced Enzyme Complementation (FEC). FEC technology employs two artificial enzyme fragments attached to separate analyte binding reagents, such as two antibodies used in a sandwich immunoassay. The two fragments are synthesized using genetic engineering techniques. When analyte binding occurs, the two enzyme fragments are forced into close proximity, resulting in enzyme activation, which can be used to drive a color reaction and generate a visual signal.

The assay can be performed in a single step, with a reaction time of about 20 minutes in a prototype assay using beta-lactamase enzyme fragments. Panbio is seeking partners to license the technology for diagnostic applications.

Another high-sensitivity homogeneous assay technology, employing DNA-Programmed Chemistry (DPC), is being developed by Ensemble Discovery (Cambridge, Massachusetts). DPC uses fluorescent label precursors attached to complementary DNA molecules, which in turn can be attached to separate binding elements such as aptamers that are specific for a target analyte.

Upon binding of both types of aptamers, the DNA molecules hybridize, resulting in assembly of active fluorescent label. The approach results in a very high effective concentration of reactants, enhancing reaction rates up to one million-fold and increasing specificity compared to the same reaction in solution.

A magnetic strategy

Dr. Richard Luxton, of the University of West England (Bristol, UK), discussed the development of a magnetic immunoassay system for rapid diagnostic applications. The system employs antibody-coated paramagnetic microparticles (PMPs) that act both as second antibodies in a sandwich immunoassay as well as labels that can be detected via magnetic sensing using a new magneto-biosensor developed by Luxton. In addition, the magnetic particles can be used to separate bound analyte, concentrating the label near the sensing surface and enhancing the signal by up to 50-fold, allowing a homogeneous assay to be performed.

Development has focused on point-of-care testing applications to take advantage of the compact size of the magneto-biosensor system, which is configured as a hand-held device. Prototype assays have been developed for C-Reactive Protein, PSA in a lateral flow format, and intracellular protein. The program has been funded in part by Randox Laboratories (Antrim, UK), a manufacturer of high-volume clinical chemistry and immunoassay testing systems and reagents, including chip-based testing devices. Another new system employing magnetic labeling is being developed by Diagnostic Biosensors (Minneapolis, Minnesota).

As discussed by Mark Tondra, PhD, of Diagnostic Biosensors, magnetic labels have the advantage of low background, since there are no magnetic materials in blood or serum samples, and high sensitivity can be achieved since existing technologies, such as magnetic readers employed in hard disk drives, allow detection of single magnetic particles with a diameter as small as one micron. Tondra has constructed a system employing a flow-through channel with a magnetic detector that can be used for cell counting and rare cell detection including detection of circulating tumor cells.

Another magnetic immunoassay format suitable for point-of-care testing applications was described at the Oak Ridge Conference by Brendan O’Farrell, PhD of Diagnostic Consulting and BioDot (Irvine, California). BioDot is the leading supplier of equipment used to fabricate lateral flow immunoassay devices, with an 80% market share. As shown in Table 3, the market for point-of-care testing products, including lateral flow immunoassay products, is approximately $3 billion worldwide, and growing in excess of 10% annually.

O’Farrell described a new Magnetic Immunochromatography (MICT) assay under development by MagnaBiosciences (San Diego, California) that employs paramagnetic particle labels and has a sensitivity that is 10-1,000 times higher than visually read immunochromatographic assays. High sensitivity is achieved in part by the ability to sense all of the labels present in the assay. POC tests have been developed for malaria parasite, Troponin I, and progesterone precursor.

Another magnetic immunochromatographic strip assay described at the Oak Ridge Conference is being developed to detect high-risk Human Papilloma Virus infection. The current version of the assay, under development by PATH (Seattle) and Arbor Vita Corporation (Sunnyvale, California), detects HPV-E6 protein at levels of 2.6 nanograms in a model system, and will next be evaluated in clinical specimens. As opposed to existing HPV detection assays that are performed in a central lab using complex equipment, the magnetic immunochromatographic strip assay platform can be used in low-resource settings.