June 15, 2006 (Vol. 26, No. 12)

Arrays for In vitro Diagnostics, Toxicology, Dose Response, and Biomarker Discovery

The maturing microarray market is less about numbers than quality of information. As users complete their high-throughput, whole-genome array work, looking at hundreds of thousands of analytes, the need to focus on subsets of those analytes is building a market for mid- and low-density panels built around disease states or genes of interest to complement companies’ in vitro diagnostics work.

The move to multiplexing is driven by the increased speed, reduced cost, and reduced sample variability of arrays, according to Ken King, vp and general manager, Biopharma for PerkinElmer (www.perkinelmer.com). Applications include toxicology, dose response and escalation, biomarker discovery, and others, but the major trend is toward in vitro diagnostics.

Medium-density Arrays xMAP

The goal at Luminex (www.luminex.com) “is to fill the chasm between single assays and high-density arrays with clinically relevant multiplexed assays,” according to Grant Gibson, Ph.D., director of scientific alliances. With some 50 partnerships, the company is a leading player in the medium-density array segment.

Luminex’ xMAP technology is a proprietary form of flow cytometry that “multiplexes 100 tests in a single sample well, by fluorescently dying beads with 100 unique colors so each carries a unique test,” Dr. Gibson explains. The Luminex 200 bioassay system is designed for clinical tests, academic research, and drug discovery. Dr. Gibson says that the xMAP technology simplifies assays by reducing the labeling and the amount of sample required, which is important for small animal work.

Bio-Rad Laboratories (www.bio-rad.com) recently introduced an 8-micron magnetic bead for heterogeneous assays performed on the xMAP platform.

The company also launched a fully automated random access product, the BioPlex 2200, to run different chemistries on the same sample in succession. It delivers 2,200 results per hour. “Fourteen additional panels are in development for autoimmune diagnostics, infectious disease, cardiovascular disease, diabetes, toxicology, and serology, plus 70 individual assays,” says Michael Barcellos, global head of marketing. The first launch is scheduled in 2006 for autoimmune diagnostics.

R&D Systems (www.rndsystems.com) developed the Fluorkine Multianalyte Profiling (FMAP) product line on the xMAP platform for high-sensitivity multiplexing using traditional ELISA reagents. “These antibodies and proteins are benchmarked for the levels analytes ought to be detected at,” points out Frank Mortari, Ph.D., director of business development. Fully validated multiplexing panels are available for human and mouse cytokines. More focused panels for obesity research and adhesion molecule-detection launch this month.

R&D also has introduced the FMAP development kit, featuring “a generic particle coated with a good antimouse immunoglobulin, so researchers can bind their own antibodies to it,” says Dr. Mortari.

Working off the Luminex platform, Serologicals (www.serologicals.com) is integrating and enhancing the multiplex products from its Upstate and Linco divisions. “We are heavily investing in new products for xMAP multiplexing,” says Parker Cassidy, vp and general manager at Linco. New products include a high-sensitivity cytokine assay and expanded LINCOplex kits for studying metabolic disorders. Existing kits multiplex up to 30 analytes on the xMAP platform. Validated assays include human, mouse, rat, and nonhuman primate versions. Serologicals has also developed assays for less invasive samples, such as tears.

Qiagen (www.qiagen.com) developed the LiquiChip as an “open system for any xMAP application to allow customers to mix and match instrumentation and consumables from any supplier,” notes Kerstin Steinert, director R&D protein expression and proteomics. It features comprehensive instruments, applications support, and unique xMAP tools, she adds, that include analysis for 6xHis-tagged proteins, RCAT signal amplification, and analysis software. Modular cytokine and cell-signaling assays are ready to use and can be automated for setup and analysis.

Beckman Coulter(www.beckmancoulter.com) partnered with Bender MedSystems(www.bendersystems.com) to distribute FlowCytomix kits and software. The kits use 96-well plates or tubes, 4.5- and 5.5-micron beads, and five fluorescence levels to analyze up to 10 analytes per well, explains Diane Lary, product manager. The kits can discriminate individual bead populations in a fluorescence channel. Human and mouse kits are available now.

Flow Cytometry Options

BD Bioscience’s (www.bdbiosciences.com) BD Cytometric Bead Array Flex Set system is an open-platform multiplex bead array that runs on a variety of dual-laser flow cytometers. Assays for this system have been screened for compatibility or cross-reactivity with other assays in this family of human, mouse, and rat cytokines and cell-signaling proteins. According to Trent Colville, product manager, research immunoassays, the Cytometric Bead Array Flex Set can run a broad platform of cytokines and chemokines.

BD’s first quantitative bead-based immunoglobulin assay, a human immunoglobulin isotyping panel, launches this summer for classifying human immunoglobulin subclasses. “One possible application,” Colville says, “is for validating novel compounds for drug development.” Also this summer, BD will introduce CVA capture beads without antibodies, so researchers can put on their proprietary content. Line extensions are ongoing.

Planar Technology

“The human proteome is so expansive that industry is leaning more toward focused protein families or disease states,” notes Richard Pembrey, Ph.D., market segment manager, Sigma-Aldrich(www.sigmaaldrich.com). “With a multiplex assay, it is possible to profile the expression of many proteins involved in the intricate cascades of signaling pathways,” Dr. Pembrey says.

The Panorama Cell Signaling Ab Array contains 224 antibodies spotted in duplicate onto a nitrocellulose slide with two positive controls and one negative control. The other segment of the Panorama family is devoted to protein-interaction profiling, including a signal-transduction microarray and a kinase microarray.

The ArrayIt division of TeleChem International (www.telecheminternational.com) has scheduled the release of transcription factor microarrays for the third quarter of 2006. The company promises better sensitivity and dynamic range than gel shifts or ELISA assays and the ability to measure 10 transcription factors simultaneously to help researchers understand their function in the context of the whole genome.

PerkinElmer, “is using comparative genomics hybridization to look for large deformities in DNA for newborn screening,” King says. “The Neogram kit includes 30 analytes specific to newborn diseases.”

Other new PerkinElmer technologies include content for Luminex beads, a scanner for the Luminex platform, and BioExpression and Agile for biomarker discovery using mass spectrometry.

Pierce Biotechnology’s (www.piercenet.com) SearchLight Multiple Array System prints capture antibodies or peptides in nanoliter volumes on 96- or 384-well plates using a combination of chemiluminescent or infrared detection and location for identification, according to Christine Burns, Ph.D., vp and general manager.

“While chemiluminscence offers unparalleled sensitivity, additionally offering infrared detection allows many more proteins, up to about 50, per well to be quantitated in a higher-density array format. With over 200 validated array pairs for chemiluminescent detection, we are launching 24 protein/well versions for infrared detection as disease-related panels for diabetes/obesity, cardiovascular disease, inflammation, and COPD to start,” states Dr. Burns. The three-step assay scans for biomarkers and is amenable to robotics. The raw data can be archived for three months at room temperature.

Last November, Invitrogen(www.invitrogen.comintroduced NCode, a comprehensive platform for miRNA profiling and detection. The platform is validated for a total of 1,100 human, mouse, rat, zebrafish, Drosophilia, and C.elegans miRNAs, “so people can make cross-species comparisons simultaneously,” according to Peter Jozsi, senior product manager, epigenetics. It includes 144 human miRNAs, predicted by in silico research, several of which have since been validated.

The company is also touting the ProtoArray Protein Microarray platform to discover novel biomarkers associated with cancer and autoimmune disease, identify drug targets, and map disease-related pathways. Human and yeast versions each have nearly 5,000 unique, full-length proteins spotted onto nitrocellulose-coated glass slides. The ProtoArray is used for immune response biomarker profiling and screening target binding.

Imaging Systems

Kelly J. Cassutt, Ph.D., application scientist at Hamamatsu (www.hamamatsu.com), says the FDSS6000 imaging-based plate reader is “an evolution not a revolution.” It multiplexes both calcium mobilization and membrane potential cell-based assays on a 96- or 384-well plate, generating two results from the same cells.

Furthermore, two different cell lines can be dye-loaded with different fluorescent calcium detection dyes and combined to generate two independent results in the same well. Additional multiplexing assays with other dyes are in development.

For cell-based imaging, Cellomics (www.cellomics.com) offers high-content screening through its family of automated imaging systems, which includes the ArrayScan VTI HCS Reader, KineticScan HCS Reader, and cellWoRx High Content Cell Analysis System. The systems provide a complete picture of the cell for image-based analysis, with tens to hundreds of parameters for each cell, the company says.

Recently, Cellomics incorporated live cell imaging to its ArrayScan VTI HCS Reader. It also is launching high-content screening kits and expects to expand its portfolio of validated reagent kits during the next two years.

This month, Promega(www.promega.com) launches its cell viability assay, MultiTox, to simultaneously assay live and dead cells in culture in the same well. According to Pam Guthmiller, strategic marketing manager, users add one reagent directly to the wells, simplifying what otherwise is a multistep process.

“Other assays, like luminescent genetic reporter or caspase assay, can be multiplexed with this reagent,” she says. In pharma, it would be used to screen compounds and provide a confirmatory, secondary assay, reducing cell culture costs and compound use.

Recent advances in nucleic acid lateral flow (NALF) multiplexing for PCR and other amplification technologies are making it “a serious contender in the future nucleic acid test market,” according to Joanna Seal, Ph.D., senior project leader, nucleic acid division, BBInternational (www.britishbiocell.co.uk).

“Multiplexed NALF involves oligonucleotide probes and gold nanoparticle conjugation technology to simultaneously detect as little as one femtomole of each PCR amplicon in a single reaction,” Dr. Seal says. Results can be visually interpreted by unskilled operators. Simple prototypes can detect up to seven targets. Using a bi-directional housing with two longer test strips can theoretically detect up to 25 targets. Commercialization is expected by 2007.

Another U.K. company SmartBead Technologies Limited (www.smartbead.com) attaches five-digit barcoded microparticles to biomolecules for a total of 99,999 unique codes.

As Chris Savory, director of sales and marketing, elaborates, “The UltraPlex barcoded microparticles have biomolecules, typically antigens or antibodies, attached to them, one biomolecule type per unique barcode. These are supplied in a lyophilized multiplexed kit format and the user incubates the microparticles with a sample. Reacting biomolecules attach to the microparticles. The reaction is incubated with a fluorescent reporter molecule, and the results of the assay are determined using our proprietary reader, which identifies each barcode and measures the amount of fluorescence associated with it.”

By reading both the barcode and fluorescence, particle identification is permanent and not subject to photo-bleaching, thus eliminating reading errors. And, Savory says, because they are lyophilized in an accusphere format, pipetting of different bead types is eliminated before running an assay, and shelf-life, stability, and antigen binding are enhanced.

Also, SmartBead is developing a biological fingerprinting solution, based on advanced pattern recognition that will lead to megaplex assays, Savory GE Healthcare (www.gehealthcare.com) are focused bioarrays in flexible multiassay array formats, according to Hrissi Samartzidou, Ph.D., global marketing manager, gene expression. The first of GE’s catalog line of multiplexed arrays, a 16-assay panel for inflammation, is being introduced this month.

New CodeLink software is also being released this month, with normalization algorithms optimized for mid-to-low density arrays and an online tool, CodeLink iCenter, with all the information relevant to CodeLink products.

Earlier this year, Promega introduced its HaloTag technology for cell imaging and protein analysis, Guthmiller says. “A gene of interest can be placed next to the HaloTag gene, expressed as a protein fusion partner, and tracked with a multitude of ligands labeled with different fluorescent moieties and multiplexed with antibodies in live or fixed-cell imaging applications,” she says. It also can be used in immobilization applications or protein-to-protein array applications using bead- or slide-based arrays.

Biacore (www.biacore.com) relies on surface plasmon resonance (SPR) for its label-free protein-interaction analysis systems, Biacore A100 and Flexchip. Biacore A100 offers a greater degree of system automation and can analyze up to 3,800 interactions in 24 hours, according to the company. It is used for protein-interaction analysis in processes demanding higher throughput, including discovery and development for small molecule drugs.

Flexchip is used for rapid screening, ranking, and profiling of large numbers of interactions and “is used for proteomics and early-stage candidate selection in biotherapeutic development,” according to the company. Both systems provide such interaction information as kinetic rate constants and affinity constants.

High-density Arrays

Before mid-sized arrays came high-density arrays. Illumina (www.illumina.com), known for its whole-genome genotyping and work on the HAPmap project, is one of the leading players. Its Golden Gate assays at 1,536-plex on each of 96 bead-based arrays on Illumina’s microplate-compatible Sentrix Array Matrix. For higher-density applications, the company uses the Sentrix Bead Chip. Its oligo-coated 3-micron beads are assembled in etched wells, resulting in an array with 12 million features per device.

The company also introduced Sentrix HumanHap Genotyping BeadChips for genome-wide disease association research. Used with the Infinium assay, researchers can interrogate the human genome at single-base resolution. Call rates exceed 99 percent and reproducibility and accuracy approach 100 percent, according to Illumina.

Beckman Coulter expanded the GenomeLab SNPstream Genotyping System to process up to 48 SNPs in each well of a 384-well plate. “Of the thousands of SNPs found in discovery, maybe between 10 and 200 are important in elucidating a particular biological endpoint,” notes Noreen Galvin, director of automation. Analyzing them would require validating thousands of SNPs.

The SNPstream scales to between 4,000 and more than 3 million assays per day, providing a cost-effective way to interrogate a realistic number of markers in a single assay, she says. “We lay down a primer, look for a mutation, and extend it by one base. If it’s there, it will bind. Fluorescence will detect it.” Beckman Coulter recently launched a multiplexing platform for studying gene expression, the GenomeLab GeXP. This capillary electrophoresis-based system enables the multiplexed expression of up to 30 genes per well.


Late last year, Invitrogen launched the HEEBO and MEEBO (human and mouse, respectively, exonic evidence-based oligonucleotide) probes for array labeling and detection, according to Wendy Price, business area manager for gene-expression profiling and genome-expression profiling. “They are used mainly for expression profiling,” she says. Designed for Illumina’s high-density arrays, in one experiment HEEBO can interrogate nearly 49,000 data points, and MEEBO can interrogate nearly 39,000 data points, Price adds.

Although this article focuses on multiplexed arrays, they are only part of the story. The other part revolves around validating specific markers, creating better antibodies to get better information and, of course, knowing how best to analyze and use the resultant information.

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