Multiplex assays are based on single-analyte tests or low-to-midplex procedures that typically predate the rise of their multiplex versions. They may include vast numbers of analytes in a single assay and require specialized technologies or miniaturization to achieve a higher degree of parallelization. Multiplexing and related issues were the focus of CHI’s “World Pharma Congress,” which was held last month, and will be on the agenda at its “Next-Generation Sequencing Summit,” which will be held in August.
“About 1 percent of the U.S. population suffers from chronic neuropathic pain, and available treatments have numerous undesirable features,” says Michael Finley, Ph.D., who is currently assay development team leader at Merck & Co. “This unmet medical need has driven an intensive effort to identify specific pain treatments.”
For these reasons, Dr. Finley and his former colleagues at Johnson & Johnson Pharmaceutical Research & Development focused on the neuronal voltage-gated (N-type) calcium channel, an appealing molecular target for the development of novel analgesics.
The N-type calcium channel is localized in the presynaptic terminals of pain fibers in the dorsal horn of the spinal cord, substantiating its pivotal role in pain response. Previously, Dr. Finley and his coworkers engineered an HEK cell line using transduction of the appropriate gene sequences that expressed the N-type calcium channel.
This cell line can be evaluated using both a fluorescence-based calcium detection system for higher throughput, as well as an automated patch-clamp electrophysiology for higher quality, albeit at lower throughput. Using the HEK cells with known inhibitors of the N-type calcium channel, Dr. Finley demonstrated that the results between the two assays were comparable.
In a large-scale evaluation, the Finley team tested about 3,000 compounds in the calcium-imaging assay in a series of eight-point dose-titration experiments. A subset of 134 of these compounds with IC50 values <50 nM were further tested in three-point dose titration in the automated patch-clamp electrophysiology assay.
The two assays showed a modest positive correlation with an approximately tenfold right shift in potency in the automated patch-clamp assay. In order to better understand the potential in vivo efficacy of the compounds, a third assay measured release of neuropeptides from primary neuronal cultures.
Zinc-Finger Protein Array
A novel approach to the multiplexed detection of pathogens in blood has been developed by David Segal, Ph.D., associate professor at UC Davis Genome Center, in collaboration with Hsueh-Chia Chang, Ph.D., Bayer professor in the department of chemical and biomolecular engineering at the University of Notre Dame.
“Our device is built in three stages,” Dr. Segal explains. “First, the pathogens are separated and concentrated from the blood; secondly the organisms are captured using antibody microarrays; and, finally, a visual detection system using engineered zinc fingered proteins produces the diagnostic signal.”
PCR-based methods are quick and sensitive, but Dr. Segal and his coworkers were aiming for multiplexible technology for which DNA hybridization is less than ideal. So they settled on a DNA binding protein, a zinc-finger scaffold that was engineered to bind to specific target sequences. However, each finger typically recognizes only three to four nucleotides of DNA.
For this reason, the investigators employed combinatorial mutagenesis to generate tandem zinc fingered domains that have the theoretical capacity to bind to 18 contiguous pairs of DNA.
A further development in the technology is the SEER-GFP feature (sequenced-enabled reassembly), which consists of split protein domains that can reassemble into an active complex only in the presence of specific, double-stranded DNA sequences. The investigators used a split beta lactamase protein that was brought together when the ZFPs found their target DNA. With a functional enzyme in place, a fluorescent substrate will generate a color chain from yellow to red.
Since the system detects double-stranded DNA, it is unnecessary to rely on production of single-stranded molecules for detection, thus the approach is more straightforward and faster than conventional PCR technology.
“We are currently working on optimization strategies in which our engineered ZFPs will detect specific pathogen DNAs in the presence of millions of nontarget sites,” Dr. Segal adds. These improvements, which are under way, will be required to realize an actual pathogen detection scenario. Moreover, in order to realize an operational system it will be necessary to integrate the components into a lab-on-a-chip device.
Dr. Segal stresses the significance of a straightforward, point-of-care device that generates an unambiguous and rapid readout, operable by an untrained individual. This would allow diagnosis of a wide range of infectious diseases by family physicians and healthcare workers far removed from high-tech diagnostic laboratories.