December 1, 2012 (Vol. 32, No. 21)

Providing cost-saving, rapid results, multi-analyte detection and analysis, and using only a tiny amount of sample, multiplexing is positioning itself as a premier technology in areas such as molecular diagnostics, patient treatment stratification, and as an essential research tool.

The young field is also tackling problems in reproducibility, efficiency, and data analysis. Whether it utilizes beads, chips, or qPCR technology, it’s providing increasing flexibility and muscle to the field. Examples include simplifying diagnostics for autoimmune disorders, automation for multiple research assays using magnetic beads, and adapting telecommunication technology to detect wavelength shifts resulting from biological interactions on a chip.

Dysregulation of the immune system can lead to self-attack by autoimmune antibodies. This often has devastating consequences leading to diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, multiple sclerosis, etc.

“Most people do not know that up to 80% of our antibodies are directed against self antigens. Health problems arise when antibodies are not properly regulated,” says Peter Schulz-Knappe, M.D., CSO and executive vp diagnostics, Protagen.

Autoimmune disorders can be elusive to definitively identify. “Often, clinicians must order multiple types of tests to correctly diagnose such disorders. Laboratories may have 30–40 different tests that could be run, but this would require about 80 milliliters of blood from a patient.”

According to Dr. Schulz-Knappe, Protagen has been working to solve this problem. “Our company has miniaturized this analysis and now only one drop of blood is needed to run the equivalent of up to 500 microELISAs. We are utilizing the bead-based Luminex xMAP technology that can simultaneously analyze 500 analytes in a single sample.

“The polystyrene microspheres are color-coded with up to 500 unique colors. We take these and covalently attach each bead color with affinity-purified human proteins that are either well-known antigens or suspected to be novel targets of autoantibodies. We currently screen more than 6,000 human proteins that were expressed in an E. coli expression system and subsequently purified.”

Luminex’ xMAP technology analyzes the reactions using a dual laser light source that excites the internal dyes for bead recognition and simultaneous autoantibody quantification. If binding has occurred, the increase in fluorescence intensity is detected and analyzed by the software.

“We are currently using this technology to screen autoimmune clinical trial samples into responder or nonresponder categories. Although the majority of current efforts in personalized medicine have been applied to cancer treatment trials, we believe autoimmune analysis is also important.”

Dr. Schulz-Knappe says the company is utilizing a three-pronged approach. “First, we coat beads with known clinically relevant antigens. Second, we also are utilizing published but potential antigens that have not been clinically validated. Third, we are continuing to clone, express, and utilize more and more proteins from the human proteome.”

Thus far, Protagen has evaluated 7,000 patient samples for 15 different indications. “The challenges faced with the technology are membrane proteins such as receptors and ion channels. Those can be tricky proteins to produce. But, overall we expect to identify many more targets and are increasingly collaborating with partners. We see a great atmosphere and also envision many more patent applications.”

Magnetic Beads and Automation

While polystyrene bead-based multiplex immunoassays require manual washing, the use of magnetic bead arrays allows automation and higher throughput.

“Newer assays developed on Luminex xMAP technology offer magnetic beads,” says Brett Houser, product manager, Bio-Rad. “These provide a number of advantages such as easy automated washing and more reproducible results. They also offer comparability with the new Bio-Plex MAGPIX reader that uses magnetic beads exclusively and offers a smaller instrument footprint-enhanced economy, as well as a more robust assay. Our focus has been magnetic for some time, we’ve recently launched over 200 assays in the magnetic format and in the next year we plan on doubling that.”

Bio-Rad’s Bio-Plex technology makes use of multiple assays whereby capture antibodies to a specific analyte are attached to a set of beads with the same color. The second antibody to the analyte is attached to a fluorescent reporter dye label. This allows simultaneous detection of numerous analytes in the same sample. The Bio-Rad Bio-Plex system provides a readout identifying the different assays by bead colors and determines the analyte concentration by measuring the reporter dye fluorescence.

Antonio Facchiano, M.D., unit chief, Vascular Pathology Laboratory The Immaculate Dermopathic Institute (IDI-IRCCS), recently utilized Bio-Rad’s Bio-Plex Pro™ human cytokine 27-plex and mouse cytokine 23-plex panels as part of his melanoma studies.

His group is seeking to find molecules or pathways with strong antiproliferative effects in order to identify early markers of melanoma and other diseases. By comparing the sera of patients to healthy individuals, the group found molecules that were expressed differently and that may eventually serve as biomarkers of the disease.

Bio-Rad has numerous other assays. It recently released the Bio-Plex Pro cell signaling assay that detects phosphorylated and other proteins in key signaling pathways. The magnetic 34 bead-based assay (23 phosphoproteins and 11 other targets) has numerous applications that range from drug discovery to disease research.

One of the challenges facing multiplexing is the sheer amount of data generated. “We have addressed this key challenge by developing software packages such as the Bio-Plex Data Pro™ for data management. It’s important that researchers have a user-friendly software package designed to extract biologically relevant data in a way that is easy to navigate, visualize, and organize.”

Bio-Rad also recently partnered with Myriad RBM to launch hundreds of new assays. “This is an ongoing priority for us.”

Photonic Technology

Another challenge of bead-based and other assays is difficulty distinguishing crosstalk, notes Martin Gleeson, Ph.D., CSO, Genalyte. “Homogeneous assays can be limited because of accessibility and compatibility issues in that the crosstalk among screening antibodies can limit the number of samples that can be analyzed at once.”

According to Dr. Gleeson, Genalyte has developed a novel multiplexing technology that solves that issue. “We are utilizing technology that was initially developed for the telecommunications and space industries. Our instrument, the Maverick™ detection system, measures wavelength shifts that occur following biological interactions on a chip and its design eliminates the problem of crosstalk.

“It consists of a silicon chip containing rings of photonic microring sensors that are coated with a distinct set of antigens that resonate at distinctive wavelengths after binding occurs. Further, this resonance shifts proportionately to the amount of sample that is bound, allowing for a quantitative evaluation. For autoimmune assays, only 2 microliters of sample (such as plasma or blood) are required. The system is incredibly easy to use, and the results are available within 15 minutes.”

The company is utilizing the system to rapidly and simultaneously screen for a number of biological analytes such as DNA, RNA, and protein. It just released its ENA4 and ENA6 assay kits that detect several of the most common antibodies found in autoimmune connective tissue disorders such as SLE.

“As compared to traditional assays such as ELISA, these tests can deliver results in a fraction of the time and effort usually needed while maintaining equivalent or often better sensitivity. This technology provides a very flexible tool for a number of applications. We are currently developing other types of arrays with additional antigens, as well as the capability for antibody isotyping.”

According to Dr. Gleeson, the field is moving toward rapid diagnostics, where he expects the speed, ease-of-use, and flexibility of the Maverick technology will be especially useful. “Given the improvements in chip quality and ability for single use, multiplexed diagnostics are within reach.”

Genalyte’s Maverick™ detection system is based on the company’s unique approach to multiplexing that, it says, reduces or eliminates sample preparation, provides scalable multiplexing for both proteins and nucleic acids from a single small sample, and achieves excellent sensitivity.

Multiplex qPCR

Multiplexing assays can run the gamut from proteins to nucleic acids. Multiplex quantitative polymerase chain reaction (qPCR) characterizes multiple DNA or RNA targets in the same sample by combining several qPCR reactions into one. All targets are amplified together and are detected independently, since different probes can have different fluorophores yielding different spectra.

According to Greer Kaufman, Ph.D., research scientist, Southern Research Institute, although multiplexed qPCR can require more time to implement and validate, it offers a number of advantages. “Analyzing multiple targets in the same sample allows conservation of sample, monitoring samples for inhibition, as well as minimizing sample loading errors and well-to-well variation.”

Damon I. Papac, Ph.D., director of the institute’s bioanalytical sciences department, described the group’s focus on multiplex qPCR. “One of the goals of Southern Research Institute is to provide contract service for clients in which we perform biodistribution and toxicology studies for vector-based therapeutics. Currently, we are performing multiplexing qPCR studies to help the field establish criteria for qualifying and validating these methods for GLP.”

The group recently reported the results of their studies. “We sought to validate a qPCR method using Qiagen’s QuantiTect Multiplex PCR kit to quantify adenovirus serotype five vector-based therapeutics in rodent blood and tissues. We performed three runs over a three-day period to assess accuracy and precision of the assay. We also performed additional runs to assess recovery, sensitivity, stability, and matrix effect.” Matrix effect examines if different tissue types alter the readout or impact results.

Following statistical analysis, the investigators determined that internal calibrators had

Finally, they determined that the viral DNA was stable after three freeze-thaw cycles and one month storage at 70°C. Overall their study validated a qPCR method for the quantification of the hexon gene from the Ad5 vector in rodent blood and tissue using the Qiagen QuantiTect Multiplex kit.

Validating each manufacturer’s kit is important, concludes Dr. Kaufman. “Manufacturers can differ in types of buffers, matrices, and other reagents they utilize in its kits. It is important to optimize and validate each method under the same experimental conditions. Only by taking the time to do that can you be confident in your results.”

Researchers at Southern Research Institute load plates into the ABI 7900HT, the high-throughput qPCR device used in a recent study. Preclinical GLP and nonGLP bioanalytical research studies are conducted in more than 10,000 square feet of custom-designed, dedicated pharmacy and lab space.

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