Assays Galore

Courtesy of BD Biosciences Pharmingen COLOR CODING: Multiplexed bead-based assays, like BD Biosciences' Cytometric Bead Array (shown), test for multiple analytes in a single vial. The key is in the colors: one hue indicates the bead ID, and the intensity of the second, how much protein has been captured. In today's fast-paced research environment, technologies for speedy, cost-efficient analyses reign supreme. As part of this general trend, techniques for multiplexing, that is, simultaneously performing different assays on the same sample within the same reaction vessel, are on the rise. "Multiplexed bead-based assay platforms are becoming increasingly popular, because they enable a great deal of quantitative information to be obtained with significant savings in labor and sample volume ... using currently available instrumentation," says Kevin Reagan, vice president of immunology at BioSource International in Camarillo, Calif. "These approaches are of broad interest because they can be used for virtually any biologic assay based on analyte capture or binding." He adds, "One particularly hot application is their use for examining the complexity of biomarkers and their interplay." Addressable beads can be used in a variety of formats, including suspension arrays, in which probes are conjugated to mobile bead sets1 that usually sport distinctive fluorescent colors and intensities; and oligo-derivatized microspheres that serve as array elements when immobilized on the tips of optical fibers.2 Researchers developing their own multianalyte assays should be prepared to invest some time in probe selection and assay optimization. Or, if applicable, they also can make use of the marketplace's growing selection of preoptimized kits for a variety of analytes, such as cytokines, phosphoproteins, and single-nucleotide polymorphisms (SNPs). "Multiplexed bead-based assays can only be as good as the reagents being employed," cautions Greg Moore, director of business development/drug discovery for Charlottesville, Va.-based Upstate Group. "Specificity and reagent compatibility are crucial. Each antibody or other analyte-capturing reagent should not only be exquisitely specific for its target, but must also function well under conditions appropriate for all of the other components of a particular panel," he adds. ON THE MAP The xMAP™ technology (formerly LabMAP3) from Austin, Texas-based Luminex is one of the most popular multiplexing approaches, enabling researchers to perform as many as 100 assays simultaneously in a single reaction vial. Luminex offers 100 unique color-coded bead sets, each of which can be coated with a specific capture reagent, such as an antibody to interleukin-4, for instance, or a specific oligonucleotide. Quantification of the analyte requires a second color, usually from a phycoerythrin- conjugated secondary antibody a labeled DNA sequence. The company's instrumentation then reads the reaction bead-by-bead, first identifying the microsphere and then measuring how much analyte is bound to it. Currently available in three different platforms, these readers are essentially specialized flow cytometers that combine fluidics, laser optics, and advanced digital signal processing. The Luminex 100 and Luminex 100 IS systems both feature a 96-well plate handler and the same core components for fluid control and sample assay. The 100 IS system also includes a bar code reader and enhanced data analysis software. The Luminex HTS, for higher-throughput applications, loads up to eight samples simultaneously from 96- or 384-well plates. Luminex itself does not actually sell microsphere sets conjugated to specific capture reagents. Instead, the company offers conjugation-ready sets, sporting either avidin, a carboxylate group, or Toronto-based Tm Biosciences' Universal Array™ oligonucleotide adapters, to assay developers who then provide assay kits, software, and instrumentation to bench scientists. "We are moving away from directly distributing our instruments in favor of selling them through our value-adding partners. In the last few years, the number of companies offering our instruments and kits has grown considerably, as has the menu of products each partner offers," notes Grant Gibson, product- marketing manager at Luminex. One popular xMAP application measures cytokines, as researchers often want to quantify a battery of these signaling molecules simultaneously. Several Luminex partners provide cytokine assays, either individually or in combination, to detect Th1/Th2 cytokines, for instance. Partners include Bender MedSystems, Vienna, Austria; Bio-Rad Laboratories, Hercules, Calif.; BioSource International; Linco Research, St. Charles, Mo.; Qiagen, Netherlands; R&D Systems, Minneapolis, Minn.; and Upstate Group. Other protein applications exist as well. Linco Research, for instance, recently extended its LINCOplex™ line of xMAP-based assays to include kits for measuring hormones related to diabetes, obesity, and cardiovascular disease, as well as for human apolipoproteins. Assays for proteins such as apoptotic markers and specific phosphoproteins, are available from Bio-Rad, BioSource International, and Upstate Group. Both Upstate Group and Qiagen offer kits to measure serine/threonine kinase activity. Another application is SNP genotyping. Tm Biosciences' Tm 100 Universal Sequence Set™, used in Luminex's FlexMAP™ beads, consists of 100 unique 24-mer tags designed to work in a single reaction while minimizing cross-hybridization. The tags can be integrated into virtually any primer for genomic screens. "We anticipate that this approach will enable the rapid transition of many genomic assays from the research lab to clinical applications," says Jeremy Bridge-Cook, Tm's vice president of business development. The company currently offers xMAP-based kits for detecting genetic mutations relating to drug metabolism and coagulation disorders. Assays for mutations associated with cystic fibrosis are under development. Marligen Biosciences in Ijamsville, Md., offers two preconfigured multiplexed systems in its Signet™ line of xMAP-based genotyping assays: one for detecting 43 male-specific SNPs on the Y chromosome, and one for distinguishing 30 genetic variations in the hypervariable regions I and II of mitochondrial DNA. The company also will work with customers to custom-design multiplexed SNP assays. "We are currently developing additional pharmacogenomic SNP assays and are also expanding into multiplexed proteomic assays," notes Jim Lazar, Marligen's vice president of research and development. For example, the company is slated to release multiplexed assays for 10 to 20 DNA-binding proteins later this year. For users who cannot find what they're looking for off-the-shelf, many of these companies will custom-produce new assays. Radix BioSolutions of Georgetown, Texas, for instance, which specializes in custom-developed assays using the client's chosen platform. A large portion of Radix's business is currently devoted to developing xMAP assays for protein and DNA analytes. "Researchers who have developed single-analyte assays often encounter problems when converting to a multiplexed platform. We ease this transition by exploring capture-reagent selection, producing the conjugated microspheres via any of a variety of different coupling chemistries, and optimizing the reaction conditions," comments Christy Weiss, Radix's vice president of operations. Many of Luminex's partners offer reagents and kits for researchers who want to try their own hands at assay development. Upstate Group, for example, offers Luminex's microspheres derivatized with capture reagents such as biotin, glutathione, nickel, or protein A/G. Qiagen offers beads and conjugates for capturing histidine-tagged proteins. Some companies also are developing relevant software. Bio-Rad, for example, offers Luminex's instrumentation integrated with Bio-Rad's Bio-Plex software for simplified system setup, data analysis/management, and automated hardware validation/calibration. MiraiBio of Alameda, Calif., offers software for streamlining data analysis, including its MasterPlex GT program for genotyping applications. Finally, Linco Research also offers an in-house training program for those just learning about xMap. CLINICAL DIAGNOSTICS Luminex's technology is being used not only in research laboratories, but also for clinical diagnostic applications by a variety of companies: Biomedical Diagnostics, Marne-la-Vallée, France; Genaco Biomedical Products, Huntsville, Ala.; Immuno Concepts, Sacramento, Calif.; Inova Diagnostics, San Diego; Multimetrix, Regensburg, Germany; One Lambda, Canoga Park, Calif.; Orchid Diagnostics, Stamford, Conn.; and Zeus Scientific, Raritan, NJ. Courtesy of Pharmaseq YOUR ASSAY'S CALLING: PharmaSeq's microtransponder A sampling of the xMAP-based diagnostic tests available or under development includes tests for autoimmune disorders, infectious diseases, thyroid conditions, coagulation disorders, and atherosclerosis. "We specialize in developing standard diagnostic tests for proteins in sera, and have opted to use Luminex's platform because it enables multiplexed analysis in a way that is easy to routinely perform in labs," comments Oliver Larbolette, Multimetrix's head of production. This technology also is being used for genotyping applications, including forensic or familial testing for identification purposes, and human leukocyte antigen (HLA) typing for matching transplant donors and recipients. "We primarily develop genotyping assays that employ 50 to 80 analytes," says Ivan Balazs, director of research and development at Orchid Diagnostics, explaining that the company adopted Luminex's technology because it seemed to provide a relatively inexpensive, user-friendly platform for multiplexing at that level. "This system is working well for us," he continues, "and we have developed several xMAP-based products for HLA genotyping, as well as protein assays for assessing the levels of anti-HLA antibodies to complement our already well-established HLA product line." Some companies, such as Future Diagnostics in the Netherlands, specialize in the contract development of assays for other companies. "We are currently working on a 24-plex assay for a client interested in testing for novel small-molecule autoantibodies," notes George Parsons, director of business development for Future Diagnostics. SPHERES OF INFLUENCE Despite its popularity, Luminex isn't the only game in town; several companies have developed competing approaches. Foster City, Calif.-based Applied Biosystems' 8200 Cellular Detection System, for instance, is an automated, scanning macroconfocal system that employs the company's FMAT® platform3 for assaying cells or beads in standard microtiter plates. For multiplexed bead assays, the size and fluorescent intensity of the analyte-bound beads are measured without the need for a wash step. Applied Biosystems also offers microspheres precoated with goat-anti-mouse IgG, kits that enable users to label their own proteins of interest, and a selection of prelabeled proteins and peptides. Bangs Laboratories in Fisher, Ind., offers its QuantumPlex™ product line for flow cytometry-based multiplexed analyses. This system employs 10 distinct microsphere populations consisting of beads of two different sizes internally dyed with different intensities of Starfire Red™. The company offers bead sets preconjugated with the multipurpose capture probes streptavidin or goat-anti-mouse IgG, or bead sets bearing a carboxylate group. San Diego-based BD Biosciences Pharmingen's line of Cytometric Bead Array (CBA) products3 is optimized for use with the company's BD FACS™ flow cytometers, but also can be used with competitors' flow cytometers in conjunction with BD CBA software. Researchers can choose from a variety of preconfigured kits for multiplexed assays, or they can custom-design panels from a growing repertoire of targets. The BeadArray™ technology from Illumina in San Diego employs addressable beads that self-assemble into microwells etched into an array substrate, effectively generating the array at run-time. The company has developed two different platforms based on BeadArray: the microplate-compatible Sentrix™ 96 Array Matrix for high-throughput applications, and the slide-shaped Sentrix BeadChip, for lower-throughput applications. Both employ Illumina's Array of Arrays® approach, in which multiple samples can be tested per "well." The Sentrix 96 Array Matrix, for instance, employs 96 fiber-optic array bundles, each containing some 50,000 individual beads. Since the system can distinguish from between 1,520 different beads, each array can test approximately 150,000 assays simultaneously. For SNP genotyping applications, Illumina uses its GoldenGate™ assay protocol, which enables more than 1,200 multiplex levels. Quantum Dot (QDC) in Hayward, Calif., has developed a system based on its Qdot™ quantum dot conjugates. (See Quantum Leap for Quantum Dots). Citing such advantages as probe brightness, photostability, broad excitation ranges, and narrow-emission spectra, QDC is developing a product line featuring QDot-labeled beads for multiplexed assays, with applications for SNP detection, immunoassays, and more. "Qbead™ microsphere assays will enable the simultaneous measure of up to thousands of analytes detected in a high-throughput reader system," notes Andy Watson, QDC's vice president of business development. Courtesy of Luminex Corp GREEN LASER, RED LASER: Luminex xMAP technology uses colored, suspended microspheres and flow cytometry instrumentation to analyze up to 100 different biological assays in a single well. A PENCHANT FOR PARTICLES Beads aren't the only particles in the multiplexing mélange. Nanoplex Technologies, Mountain View, Calif., for example, has developed a technology for manufacturing tiny, cylindrical particles that serve as the nanoscale equivalents of conventional bar codes. Fashioned from inert metals such as gold, nickel, platinum, or silver, these Nanobarcodes™ are encoded with a series of machine-readable, submicron stripes. The diminutive rods can be complexed directly to biological molecules for various applications, including multiplexed assays. The company says that because Nanobarcodes™ are identified by virtue of their intrinsic reflectivity, an unlimited number of these uniquely coded tags could potentially be designed. Detection can be effected with any of a variety of instruments, including optical microscopes or flow cytometers, in conjunction with the requisite software for decoding and analysis. The UltraPlex™ system from SmartBead Technologies, Cambridge, UK, employs a different barcoding strategy. This company's microscale aluminum particles contain integral barcodes comprising a series of holes. Identification occurs via a binary system in which each hole and intervening segment of a specified length of perforated metal is defined as 0 and 1, respectively. More than one million unique codes are possible. UltraPlex barcodes can be used to tag biomolecules for various applications, including multiplexed assays. SmartBead's reader employs automated image processing equipment and software for decoding and analysis. Finally, Princeton, NJ- based PharmaSeq has developed a novel system for multiplexed DNA analyses. Appropriate oligonucleotide probes are coupled to sets of light-activated microtransponders, each broadcasting a unique identification number via radio signal. After hybridization with the fluorescently labeled sample nucleic acid, a scanner consisting of a high-speed flow fluorometer modified to detect radio frequency is used to read the fluorescent signal as well as the unique identification number associated with each microtransponder. Lanette Fee and Deborah A. Fitzgerald can be reached via the contact form posted at www.SciWriters.com. References 1. J.P. Nolan, L.A. Sklar, "Suspension array technology: Evolution of the flat- array paradigm," Trends Biotechnol, 20:9-12, 2002. 2. J.A. Ferguson et al., "High-density fiber-optic DNA random microsphere array," Anal Chem, 72:5618-24, 2000. 3. D.A. Fitzgerald, "Assays by the score," The Scientist, 15[11]:25, May 28, 2001. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Assays Galore

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