Assays by the Score

Click to view the PDF file: Bead-based Fluorescent Multiplex Protein Analysis Systems Courtesy of LINCO ResearchLabMAP-based systems use internally dyed fluorescent microspheres to analyze as many as 100 different analytes concurrently. Today's competitive, high-paced research environment has stimulated the development of a host of approaches for rapid, cost-efficient analyses of large numbers of samples. In keeping with this trend, methods for simultaneously analyzing multiple species in a given sample have also been devised. Multiplexed assays are now a mainstay of the DNA world. For example, real-time PCR and in situ hybridization techniques can be multiplexed through the use of multiple fluorophores. Although not as well known, methods for simultaneously assaying different proteins in individual samples are also on the market. This overview highlights several commercially available systems for multiplexed protein analyses, with a focus on bead-based methodologies that employ fluorescent detection methods. The versatile laboratory multianalyte profiling (LabMAP™) system developed by Luminex Corp. of Austin, Texas, can be used for virtually any bioassay that is based on the specific binding of one molecule to another.1-3 Applications include assays for nucleic acids, antigen/antibody binding, receptor/ligand binding, and enzyme activity. LabMAP assays for enzyme activity can be based on the immunological detection of the product, or, in some cases, may follow the gain or loss of fluorescence (e.g., in protease-catalyzed cleavages of fluorescently labeled peptide targets). LabMAP assays employ three different fluorochromes: two to create color-coded microspheres, and the third for quantifying the reaction. Polystyrene microspheres are internally dyed with precise ratios of two spectrally distinct fluorochromes. This ratio confers a unique identifying "signature" or "spectral address" to each microsphere set. Bioassays are conducted on the surfaces of the microspheres. Each capture probe (e.g., analyte-specific antibody or complementary oligonucleotide) is immobilized onto a color-coded set of microspheres using any of a variety of different surface chemistries. Luminex offers microspheres bearing Lumavidin™ (an avidin derivative), for immobilizing biotinylated molecules, or carboxyl groups, for covalently coupling protein or DNA. The binding of analyte to an immobilized probe is detected via a detection reagent labeled with the third fluorochrome. Luminex currently offers 100 different microsphere sets, each of which can be used for the simultaneous measurement of a different analyte. Thus, in theory, up to 100 different species can be simultaneously measured in a single tube or microplate well. "Generally, I have been very impressed with the flexibility, multiplexing capability, and future potential of the Luminex system. Fluorescent beads may become a major platform for assay development in the next decade," comments Dario Vignali, associate member of St. Jude Children's Research Hospital in Memphis, Tenn. The microspheres employed in an earlier version of this technology (FlowMetrix™) were designed for use with a flow cytometer. This product line has been phased out, and the second-generation product line is designed specifically to work with the instruments available from Luminex or their partners. The Luminex 100™ instrument uses microfluidics to align the microspheres in single file and employs two lasers, one for the detection of the fluorescent microsphere itself, and the other for the reporter reagent. The color signals are captured by an optics system and translated into binding data via digital signal processing. A microplate interface that offers walk-away operation and robotic capability and a component that allows continuous, unattended sample delivery are also available. The Luminex HTS™ system was designed for high-throughput analyses, and may be used to perform roughly 400,000 assays per day. MAPping Out Instrumentation, reagents, and custom services for LabMAP technology users are also available from companies other than Luminex. For example, Bio-Rad Laboratories of Hercules, Calif., introduced the Bio-Plex™ Protein Array System at the December 2000 meeting of the American Society for Cell Biology. This fully integrated system combines a fluorescent reader with the software, protocols, and supplies needed for performing LabMAP-based assays in a 96-well microplate format. The company will initially offer kits for single or multiplex assay of up to eight cytokines, including several interleukins, GM-CSF, IFN-g, and TNF-a. Bio-Rad also offers kits for simultaneously measuring the phosphorylation levels of any or all of the following protein targets: JNK/SAPK1, p38 MAPK/SAPK2, p24 MAPK/Erk2, IkB-a, and AKT1/PKBa. "The primary benefits of the Bio-Plex Protein Array System, other than up to a 100-fold increase in data, include significantly reduced sample requirements, and savings in assay time, labor, and expenses," comments Erik Willis, marketing manager for the Bio-Plex product line. The system's software offers numerous features, including automated generation of multiple standard curves, four-parameter logistic curve fitting, automated system monitoring and error flags, sorting and screening tools for rapid sample analyses, and a standard exportable data file format. Upstate Biotechnology of Lake Placid, New York, offers the Luminex 100 instrument as well their Beadlyte™ line of assay kits and reagents, which is based on the LabMAP technology. The company offers kits for the multiplex detection for up to ten human cytokines (IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, GM-CSF, IFN-g, and TNF-a), or up to nine of the murine counterparts (IL-1b, IL-2, IL-4, IL-6, IL-10, IL-12, GM-CSF, IFN-g, and TNF-a). Upstate Biotechnology also offers a wide variety of ancillary and "general-purpose" reagents, including microspheres labeled with capture probes that specifically bind avidin (biotin), immunoglobulins (secondary antibodies and protein A/G), or common fusion protein tags (Ni2+ and glutathione). The company currently offers a Beadlyte assay kit for the detection of a wide variety of tyrosine kinases,4 and will soon introduce a kit for the simultaneous detection of serine, threonine, and tyrosine kinase activities. This kit will employ three bead sets that contain specific substrate peptides for their respective kinases. The reaction is detected using biotinylated phosphoserine, phosphothreonine, and phosphotyrosine monoclonal antibodies followed by streptavidin-phycoerythrin. "We think that this kit will be helpful for drug discovery studies because it can be used to distinguish between specific kinase inhibitors and ones that nonspecifically block ATP binding to a kinase," explains Greg Moore, drug discovery product manager at Upstate Biotechnology. "Many HTS kinase studies identify compounds that competitively inhibit ATP-binding, and there is a need for secondary assays to separate them from potential lead compounds," adds Moore. Upstate Biotechnology also offers its Bead-link™ custom conjugation services, in which the researcher's protein of interest is linked, via proprietary methods, to the fluorescent microspheres. According to the company, their Bead-link methodologies increase fluorescent signals and lower background noise. Courtesy of LINCO ResearchLINCO Research's LINCOplex human cytokine multiplex immunoassay kit LINCO Research Inc. of St. Charles, Mo., offers LabMAP-based multiplex immunoassay kits under the trade name LINCOplex™. The company currently offers kits for the multiplexed detection of up to eight human cytokines (IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-g, and TNF-a); kits for IL-12 and GMCSF will be available soon. LINCO Research will offer an assay kit for simultaneously measuring mouse cytokines and murine/human endocrine hormones (C-peptide, glucagons, insulin, and leptin) by mid-2001. Biosource International of Camarillo, Calif., also offers kits based on the Luminex 100 system. Biosource offers both antibody-pair bead kits and separate buffer kits. The bead/detector antibody pairs may be purchased separately, to allow for maximum flexibility in experimental design. Current offerings include a Th1/Th2-specific cytokines kit (detecting IL-2, IL-4, IL-5, IL-10, and IFN-g) and a sepsis/inflammation kit (detecting IL-1b, IL-6, hIL-8, TNF-a, and MIP-2, a murine IL-8 homolog). Future offerings will focus on innate immunity, neuroscience, and signal transduction markers. Earlier this year, Biosource International entered into a strategic partnership with MiraiBio Inc. of Alameda, Calif., formerly the Genetic Systems Division of Hitachi Software Engineering America Ltd., to market Luminex systems, reagents, and software. Under this agreement, MiraiBio will offer Luminex systems and customized software to analyze the data from LabMAP assays. Biosource will sell LabMAP reagent kits worldwide, except in Japan, where MiraiBio will sell kits co-branded with Biosource. Flowing Forward The Cytometric Bead Array (CBA) system from BD Biosciences of San Diego is flexible in that it accommodates multiple sizes and fluorescent intensities of particles. The system includes everything the researcher needs to implement this technology, including a cytometer setup kit with the requisite software, reagents and standards. The company's CBA assay kits employ their proprietary bead sets, which are internally dyed with varying intensities of a proprietary fluorophore. These sets are distinguished via one fluorescence parameter and two size discriminators. However, the system is also capable of handling assays based on the use of other types of spectrally distinct microsphere sets. The CBA analysis software is an "add-in" for Microsoft Excel®, and is compatible with contemporary data acquisition software such as CellQuest™. Researchers can employ a variety of preset configurations for generating standard dilution series and calibration curves, and data reports can be generated at each step in the process. "One advantage of our CBA system is that it employs a flow cytometer, an instrument already installed in many laboratories, for detection," comments Tony Ward, marketing director of BD Biosciences. The company currently offers CBA kits for isotyping mouse immunoglobulins and for the multiplexed detection of cytokines from human (IL-2, IL-4, IL-5, IL-10, IFN-g, and TNF-a) or mouse (IL-2, IL-4, IL-5, IFN-g, and TNF-a) sources. Several additional kits relating to inflammation are currently in development. "Developing multiplexed assays is often very challenging because each individual reaction has its own optimal conditions. By taking advantage of 'ready-to-use' kits such as ours, researchers may be able to spare themselves considerable time and effort," adds Ward. Bangs Laboratories Inc. of Indianapolis introduced its QuantumPlex™ line of products early this year. The company currently offers kits of bead sets bearing either of two "general purpose" capture probes: goat anti-mouse IgG or streptavidin. Carboxylated bead sets will be available this summer. Beads are available in two different sizes, 4.4 micron or 5.5 micron. For both of these sizes, there are five spectrally distinct bead populations. These are internally dyed with varying intensities of Bangs Laboratories' proprietary dye, Starfire Red™. The systems discussed up to this point employ microspheres internally dyed with one or more fluorophores. BioErgonomics Inc. (BioE™) of Saint Paul, Minn., takes a different approach. This company offers cytokine-specific, antibody-coated paramagnetic beads that capture the cytokine present in a sample.5 The captured cytokine is then detected using a fluorochrome-labeled, anti-cytokine monoclonal antibody. The concentration of the cytokine in the original sample is directly proportional to the intensity of the resultant fluorescent signal. Because the capture particles have significantly different light-scatter properties than cells, they do not have to be separated from the fluid phase during the experiment but can be separated spatially on light-scatter histograms. However, these particles may be recovered afterward via centrifugation or magnetic attraction. The cytokine immunoassay kits are currently available in formats for the detection of single or multiple analytes. The multiplexed MultiFlow™ assays may be used to simultaneously quantify three different cytokines using the fluorescein, PE, and cy5PE emission ranges of detection. Three different MultiFlow kits, each for the assay of different sets of human cytokines, are currently available. Assay kits for mouse cytokines will be offered in the near future. "We are continually expanding our base of cytokine assay kits, and are moving into other areas of analysis such as hormones, tumor markers, and infectious disease detection," remarks Daniel Collins, chief scientific officer at BioE. MultiFlow assays employ standard flow cytometry practices, and do not require specialized software or instrumentation. "Many therapeutic targets studied by major pharmaceutical and biotechnology companies impact the immune system. Our MultiFlow assays for cytokines enable researchers to rapidly and cost-effectively screen the immune response to potential therapeutic targets, and may thereby accelerate the rate of new drug discovery," comments Collins. Scanning Along Courtesy of Applied BiosystemsApplied Biosystems' FMAT system 8100 The FMAT™ System 8100 from Applied Biosystems of Foster City, Calif., is an automated, macro-confocal system for multiplexed, fluorescence-linked immunosorbent assays (FLISA).6,7 This system employs fluorometric microvolume assay technology (FMAT™), and was developed by Applied Biosystems in collaboration with Biometric Imaging, a BD Biosciences subsidiary now based in San Jose, Calif. The FMAT System 8100 incorporates a laser scanner and an optical detection system. The complete system includes an instrument integrated with robotic plate-loading capability, and a computer with intuitive analysis software that formats data for easy integration into various information management systems. Applied Biosciences also offers custom labeling services, and will provide training for its customers. During analyses, the FMAT System's helium/neon laser scans across microplate wells, exciting fluorescently labeled target beads (or cells). The laser objective collects emitted fluorescent signals, and the software interprets these signals as three-dimensional images that represent cells classified by their shape, size, fluorescent intensity, and wavelength. According to Applied Biosystems, its FMAT-based FLISA can be easily adapted from existing immunoassays and is comparable to traditional ELISAs with respect to linear dynamic range and sensitivity. Because the system is capable of recognizing and ignoring background fluorescence, no wash steps are required to remove unbound antibody, ligand, or fluorophore. The instrument can detect two different fluorescent dyes, and fluorescent beads of various sizes can be distinguished from one another. Thus, it is capable of multiplexed assays that employ bead sets discernable via color and/or size. This system was designed for high throughput screening applications; assays may be performed in 96-, 384-, or 1536-well plates. Deborah Fitzgerald (df@sciwriters.com) is a freelance writer in Birmingham, Ala. References 1. R. Carson, D. Vignali, "Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay," Journal of Immunological Methods, 227:41-52, 1999. 2. D. Vignali, "Multiplexed particle-based flow cytometric assays," Journal of Immunological Methods, 243:243-55, Sept. 21, 2000. 3. M. Spain, R. McDade, "A workstation approach to bioassays," IVD Technology, 07:35, 2000. (www.devicelink.com/ivdt /archive/00/07/004.html) 4. D. Fitzgerald, "Escaping the heat," The Scientist, 14[22]:23, Nov. 13, 2000. 5. D.P. Collins, "Cytokine and cytokine receptor expression as a biological indicator of immune activation: important considerations in the development of in vitro model systems," Journal of Immunological Methods, 243:125-45, Sept. 21, 2000. 6. E. Swartzman et al., "A homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology," Analytical Biochemistry, 271:143-51, 1999. 7. S. Miraglia et al., "Advances in technology to improve HTS - Developing multiplexed assays," In High Throughput Screening - The Next Generation, G. Dixon, J. Major, M. Rice (Eds.), Boston Scientific Publishers Ltd., Oxford, UK, pages 11-20, 2000.

Assays by the Score

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