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Multiplexed microsphere-based flow cytometric assays
Experimental Hematology 30 (2002) 1227–1237
Multiplexed microsphere-based flow cytometric assays Kathryn L. Kellara and Marie A. Iannoneb a National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Ga., USA; bGlaxoSmithKline, Research Triangle Park, NC, USA
Flow cytometry has become an indispensable tool for clinical diagnostics and basic research. Although primarily designed for cellular analysis, flow cytometers can detect any particles in the lower micron range, including inert microspheres of different sizes, dyed with various fluorochromes. Over the past 20 years, microspheres have been used as calibrators for flow cytometers and also as a solid support for numerous molecular reactions quantitated by flow cytometry. Proteins, oligonucleotides, polysaccharides, lipids, or small peptides have been adsorbed or chemically coupled to the surface of microspheres to capture analytes that are subsequently measured by a fluorochrome-conjugated detection molecule. More recently, assays for similar analytes have been multiplexed, or analyzed in the same assay volume, by performing each reaction on a set of microspheres that are dyed to different fluorescent intensities and, therefore, are spectrally distinct. Some recent applications with fluorescent microspheres have included cytokine quantitation, single nucleotide polymorphism genotyping, phosphorylated protein detection, and characterization of the molecular interactions of nuclear receptors. The speed, sensitivity, and accuracy of flow cytometric detection of multiple binding events measured in the same small volume have the potential to replace many clinical diagnostic and research methods and deliver data on hundreds of analytes simultaneously. © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.
Flow cytometry has been used for the multiparametric analysis of hematologic and immunologic cells for over 20 years. Although the majority of the applications are cell based, particles such as microspheres can also be detected by laser light scatter. Microspheres have a considerable history as the solid support for a variety of molecular reactions that are analyzed by flow cytometry. As early as 1977, flow cytometry was applied to the detection of antigens captured by antibodies coated on the surface of microspheres [1]. In addition, polymerase chain reaction (PCR) products have been detected by hybridization to oligonucleotide probes bound to microspheres and enzymatic reaction products have been captured on microspheres [2,3]. These basic molecular applications have been expanded to the simultaneous detection of multiple analytes in the same sample volume by combining individual reactions on spectrally distinct sets of microspheres. This review will explore the expansion of flow cytometry to the detection of these multiplexed molecular reactions on an array of fluorescent microspheres.
Offprint requests to: Kathryn L. Kellar, Ph.D., CDC/NCID/SRP, MS D-34, 1600 Clifton Rd., NE, Atlanta, GA 30333 USA; E-mail: klk@ cdc.gov
Microsphere technology Historical perspective Antibodies and antigens have been adsorbed or directly or indirectly coupled to microspheres composed of a variety of materials, such as latex, polystyrene, polyacrylamide, and glass [4–7]. Immunoassays based on the physical adsorption of antibodies to microspheres for analyte quantitation include ones for -2 microglobulin [8], Clostridium difficile toxin [5], and -fetoprotein [9]. Human IgG [6] and IgE [10] have been measured with antibodies chemically coupled to microspheres. Likewise, antigens have been adsorbed to microspheres to detect antibodies specific for Helicobacter pylori [11], human IgA [12], light chains [13], and phospholipids [14–16] or covalently attached to detect antibodies to -gliadin [17]. Microspheres coated with human C1q were used to detect circulating immune complexes containing elusive HIV antigens [18,19] and collagen has been used to capture vWF [7]. Although flow cytometric detection of PCR and enzymatic reaction products captured on microspheres was not widely employed until recently, there were a few early reports. The activity of potential inhibitors of gelatinase B was monitored by the fluorescence of substrates adsorbed to microspheres [3]. Flow cytometric detection of fluorescein-
0301-472X/02 $–see front matter. Copyright © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(02)0 0 9 2 2 - 0
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ated PCR products captured on oligonucleotide- or streptavidin-coated magnetic beads was shown to be more sensitive and less hazardous than traditional methods, such as dye- or radioisotope-resolved gel electrophoresis or chemiluminescence of filter-hybridized products [2,20,21]. The unique properties of microspheres, which include their range of sizes, stability, uniformity, and capacity to absorb and retain fluorescent dye, were readily adapted to flow cytometric analysis of multiplexed reactions. Some of the first multiplexed immunoassays detected antibodies to cytomegalovirus and herpes simplex virus [22], different Candida albicans preparations [23], hepatitis C core and nonstructural proteins [24], and four recombinant DNA-produced HIV proteins [25] after capture on antigen-coated microspheres of different sizes.
charides can be purchased. Noncovalent linkages are useful for some applications. Species-specific anti-IgGs coupled to microspheres facilitate direct binding of immunoglobulins from ascites fluids or concentrated hybridoma supernatants when further purification is not practical. High- and lowdensity streptavidin-coated fluorospheres with the capacity to bind biotinylated analytes are available for adjusting the dynamic range of quantitative assays and signal-to-noise ratios (Radix Biosolutions, Georgetown, TX, USA). Similarly, other affinity tags such as glutathione-GST, Ni-6xhistidine, and protein A and G can be utilized to link capture proteins to microspheres (Upstate Biotechnology, Waltham, MA, USA; QIAGEN Sciences, Valencia, CA, USA). Procedures for covalent and noncovalent protein coupling to microspheres have been reviewed previously [26].
Microsphere availability Microspheres of various sizes and composed of different polymers are available from a number of vendors (Table 1). Several companies offer an “array” of fluorescent microspheres with different emission spectra and intensities that permit the simultaneous analysis of several to potentially a hundred analytes. Fluorescent microspheres with carboxyl, amine/hydrazide, and maleimide groups for covalent coupling of proteins, peptides, oligonucleotides, and polysac-
Instrumentation Conventional flow cytometers with the appropriate lasers for the respective fluorochromes can acquire microspherebased emission data. In the mid-1990s, the Luminex Corporation (Austin, TX, USA) coupled a digital signal processor and a Becton-Dickinson benchtop cytometer equipped with a 488-nm argon laser to detect and process thousands of fluorescent signals generated by multiplexed microspherebased assays in real time (FlowMetrix System) [27]. With
Table 1. Resources for multiplexed microsphere-based flow cytometric assays* Source†
Web site
Instrumentation, software, and kits
Applied Cytometry Systems BD Biosciences Bio-Rad Laboratories MiraiBio, Inc. Luminex Corp.
appliedcytometry.biz bdbiosciences.com/pharmingen bio-rad.com miraibio.com luminexcorp.com
Microspheres
Bangs Labs Duke Scientific Luminex Corp. Molecular Probes Polysciences, Inc. Qiagen Radix BioSolutions Seradyn Spherotech Upstate Biotechnology
bangslabs.com dukescientific.com luminexcorp.com molecularprobes.com polysciences.com qiagen.com radixbiosolutions.com seradyn.com spherotech.com upstatebiotech.com
Multiplexed immunoassay kits
Bender MedSystems BioErgonomics Biosource International LINCO Research, Inc. One Lambda R & D Systems Upstate Biotechnology Zeus Scientific, Inc.
bendermedsystems.com bioe.com biosource.com lincoresearch.com onelambda.com rndsystems.com upstatebiotech.com zeusscientific.com
Software
Brendan Scientific
brendan.com
Resource
*This list includes some of the companies that have products for multiplexed fluorescent microsphere-based assays † The use of trade names and commercial sources is for identification purposes only and does not imply endorsement by the Centers for Disease Control and Prevention or the Department of Health and Human Services.
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this system, 64 sets of microspheres dyed with different intensities of two fluorophores were available for multiplexed assay development [28]. A disadvantage of the FlowMetrix system was the need to compensate for the overlap in fluorescence between the reporter (FL1 fluorescence) and the microspheres (FL2/FL3 fluorescence). This system was replaced with an instrument specifically designed to analyze fluorescent microsphere-based assays. The Luminex 100 and proprietary microspheres were developed to provide a high signal-to-noise ratio for detection of low-level fluorescence with PE as the reporter (Luminex Corp.). The Luminex 100 is a compact benchtop cytometer equipped with a digital signal processor and two inexpensive lasers. The 532-nm, 13-mW yttrium aluminum garnet (YAG) laser was selected for improved excitation of R-phycoerythrin (PE, 578-nm emission) compared with a 488-nm laser. The 635-nm 10-mW red diode laser excites the two classification fluorochromes embedded within the proprietary 5.6-m microspheres. Different ratios of these dyes define 100-microsphere populations. The dyes emit at 658 and 712 nm and have no spectral overlap with reporters such as PE or Alexa 532, so no fluorescent compensation is required [29,30]. An optional X-Y platform for reading 96-well microtiter plates facilitates automated sample acquisition. With this platform, both hardbottom and filter-bottom “wash” plates (Millipore Multiscreen, Burlington, MA, USA) can be processed.
Multiplexed microsphere applications Immunoassays Multiple immunoassays can be performed simultaneously on an array of microspheres as long as the conditions of the assays (incubation times, sequence of reagent additions, washes) are the same or can be combined. For quantitative sandwich immunoassays, standard curves are optimized one analyte at a time, with the capture antibody coupled to the microsphere and the detection antibody bound to the reporter (Fig. 1). The general principles of ELISA development, which are to titer each reagent and modify incubation times, temperatures, and washing conditions, are applicable. Often microsphere-based assays can be performed with no wash steps if the background and potential interfering substances in the samples are minimal. They can also be done in one step [31,32]. Since each microsphere population is distinguished by size and/or spectral address, the reporter intensity relates to the quantity of analyte bound and thus establishes the relative concentration of that analyte. Commercial antibody pairs optimal for ELISA development may not always be optimal for multiplexed microsphere immunoassays. Antibodies should be tested to find the best pairs, be matched for affinity, and not be reactive with the same epitope [33]. The most stringent assay conditions for the weakest pair of antibodies define the conditions of the multiplexed assay. Once optimized, the individual as-
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says can be combined into the reaction volumes of one assay. The cross-reactivity of all analytes and antibodies in a multiplexed format must be minimal. A primary difficulty encountered with multiplexed immunoassays is the false-positive or -negative results from heterophilic antibodies, human anti-animal antibodies, rheumatoid factor (RF), soluble receptors for the analyte, etc., similar to those observed with human serum or plasma samples in two-site assays such as ELISAs [34–39]. Multiple antibody pairs in a multiplexed format compound the potential for interference. For these assays to be effective, the standards and blanks should be diluted in a matrix similar to the samples, i.e., cell culture supernatants, serum, etc., and blocking agents similar to those used for ELISAs should be added [40]. Fixation with a final 1% formaldehyde or similar fixative is often necessary to ensure that reaction times are the same for standards and samples that may be read over the span of 30 to 60 minutes on a cytometer [40]. Fixation also inactivates any potential biohazards in human body fluids that fall under the guidelines of the OSHA Regulations for Bloodborne Pathogens (osha.gov/OSHStd_data/1910_1030. html). Unfixed human samples should not be read directly on instruments in the open laboratory. The steps from setup to data analysis of a microspherebased immunoasssay are outlined in Figure 1. An antibody detection and a sandwich immunoassay format are diagrammed, but any immunoassay may be designed. The fluorescent intensities of each fluorochrome are measured and, if quantitative, the assay data are transformed with analytical software. In this example, the microsphere populations are classified with two fluorochromes and a third fluorescent reporter, PE, is used to quantitate eight cytokine concentrations. The 8 data points for each sample are derived from a minimum of 2400 signals (8 populations of microspheres, 100 microspheres acquired per population, 3 fluorescent signals per microsphere). Although the speed of multiplexed assay performance and data acquisition is a definite advantage, the speed of data analysis is just as critical for high-throughput data processing. If an array of 100 sets of microspheres is set up in each of 96 wells, a minimum of 2,880,000 signals need to be condensed into a manageable data output. Several analytical software packages that perform statistical curve fitting and compute analyte concentrations in real time are currently available (Table 1) [32,41]. Quantitation of cytokines Cytokines, chemokines, and growth factors (hereafter referred to collectively as cytokines) are excellent candidates for multiplexed analysis because of their complex interrelationships in immunologic and hematologic functions. Fluctuations in the level of one cytokine often induce changes in others. Thus, the rates of secretion of these analytes in a variety of physiological conditions are best measured simultaneously in identical reaction formats.
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Figure 1. Steps in a multiplexed microsphere-based flow cytometric immunoassay. (A) Antibody detection and sandwich immunoassay formats performed on microspheres in liquid suspension are diagrammed. Any antigen-antibody-based format can be designed with a terminal reporter molecule specific to the laser detection system and distinct from any fluorescence of the microspheres. (B) At the completion of the binding assay, fluorescent intensities associated with each microsphere are measured by flow cytometry. In this example, the optical detection system of the Luminex 100 is illustrated (see text). (C) The dot plot displays 100 distinct regions for each Luminex microsphere population classified on the basis of the intensities of two internal fluorochromes. The instrument was set to acquire at least 100 microspheres each of 8 spectrally distinct populations of microspheres. (D) Multiplexed data from a random sample in an 8-plex assay was calculated from the log-log transformation of the values for the standards using SOFTmax PRO software (Molecular Devices, Sunnyvale, CA, USA) and graphed with Excel 2000 software (Microsoft Corp., Redmond, WA, USA).
Multiplexed microsphere arrays for cytokines developed by commercial vendors or independent laboratories are primarily two-site immunoassays. Relatively high-affinity matched pairs of antibodies for ELISAs for at least 50 human cytokines and related molecules are available from commercial vendors. The limit of sensitivity of these commercial antibody pairs falls in the low picogram range (R & D Systems, Inc., Minneapolis, MN, USA; Pharmingen/BD Biosciences, San Jose, CA, USA; among others). This limit derives from high-affinity antibodies (KD 108 to 1010) that, in combination with liquid-phase kinetics and the high binding capacity of three-dimensional microspheres, produce a robust multiplexed immunoassay. The full range of these assays extends over 3 to 4 logs, as compared with 1 to 2 logs for ELISAs. A multiplexed array for 15 mouse cytokines is the largest described to date [33]. The FlowMetrix system was employed to study the secretion of TH1 vs TH2 cytokines from
stimulated mouse CD4 T cells. Overall patterns of cytokine secretion were similar with either microsphere or ELISA methods. However, the FlowMetrix assay had a lower limit of sensitivity because a reduction in the number of microspheres reacted per cytokine and extension of the incubation times increased the reporter signals. The multiplexed assay was also more reproducible. The data were derived from measurements of at least 100 microspheres per cytokine, with each microsphere functioning as an independent assay. These measurements were more reliable than a single reading from the bottom of a microtiter well for ELISAs [33]. Multiplexed assays for up to nine human cytokines have been described [31,32,40–43]. Different combinations of interleukins (IL)-1, -2, -4, -5, -6, -8, -10, -12, TNF-, IFN-, GM-CSF, IFN-inducible protein (IP)-10, and RANTES were measured in whole blood, cell-culture supernatants, tears, and serum. The performance of the multiplexed formats for human cytokines has been validated by compari-
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sons with ELISAs [31,32,40–42]. Based on the historical variability of results obtained with different ELISA kits for the same antigen, it is fair to say that comparisons between multiplexed assays and ELISAs are most valid if the same antibodies and cytokine standards are used in both formats. Such comparisons demonstrated that the detection limits were the same or better for the multiplexed assays [31, 32,40–42]. Sample data from the two methods were highly comparable (correlation coefficients 0.857 to 1.0) [31,32,40,42]. Reproducibility was excellent for the multiplexed format: intraassay CVs were less than 10% [31,41] and interassay CVs were less than 5% [41]. Recoveries of 8 cytokines spiked into a buffer or normal serum matrix averaged 102.5 5% (n 13) [40]. Microsphere-based arrays are more rapid, less expensive, and require less sample volume than ELISAs [32,33,40– 42,44]. In the near-liquid phase of the former, equilibrium is reached sooner so incubation times are reduced significantly, particularly if wash steps are eliminated. Based on an assay for 8 cytokines (8-plex), Kellar and colleagues calculated that 8 ELISA kits cost 17 times that of multiplex reagents (Kellar, unpublished observations) and the 8-plex required less than or equal to 1/20th the amount of sample (20 L vs 400 L for ELISAs) [40]. Other methods for measuring cytokine production are ELISPOT and intracellular staining [45,46]. Neither technique is quantitative; both measure the frequency of cells producing detectable levels of specific cytokines. The ELISPOT technique detects the secretion of a specific cytokine from individual cells deposited in the wells of a multi-well plate. The wells are surface-coated with antibody and subsequently developed for a colorimetric reaction, similarly to ELISAs. The number of cytokine-producing cells is more often manually enumerated [45]. Intracellular cytokine detection by flow cytometry also measures cytokine production by single cells. Intracellular detection is accomplished by fixation and permeabilization of cells followed by specific antibody staining. With this technique, multiple cytokines as well as cell types can be identified by multiparametric analysis of a heterogeneous cell population [46]. By combining various techniques such as ELISPOT or intracellular staining with microsphere-based assays to quantitate secreted cytokine and methods that measure cytokine receptors, a comprehensive analysis of the regulation of cytokine production can be made [47].
Analytes in newborn blood spots A microsphere-based test for congenital hypothyroidism has been reported [48]. Proteins eluted from dried blood spots were analyzed by combining two microsphere assays, one a competitive assay for thyroxine (T4) and the other a sandwich immunoassay for thyrotropin (TSH). Similarly, HIV-1 antigens on microspheres were used to detect HIV antibodies [49].
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Viral and bacterial identification Two groups have reported bacterial serotyping of multiple strains of Streptococcus pneumoniae [50,51]. Both assays were based on microspheres coated with strain-specific polysaccharides and binding of serum IgG antibodies, followed by a fluorescent secondary antibody. A measure of viral load was developed by covalently coupling adenovirus antibodies to microspheres and incubating with DNAspecific fluorophores [52]. The mean fluorescence intensity was measured from a standard curve of fluorescent intensity vs viral concentration. The identification of bacteria and viruses by DNA-based methods is described in the single nucleotide polymorphism genotyping section of this review. Fluorosphere-based analyses would be applicable for disease surveillance [53], vaccine development and surveillance, screening of hybridomas and donated blood, and other highthroughput screening procedures. Jani et al. [53] have proposed that multiplexed immunoassays for sexually transmitted diseases, childhood diseases presenting with rash and fever, or testing critical to blood banking could be performed on a small, inexpensive flow cytometer placed in the underserved regions of the world for healthcare delivery and disease surveillance.
HLA testing Flow cytometry has significantly impacted HLA testing [54,55]. Microspheres coated with HLA class I and class II antigens have been used to test for HLA antibodies prior to transplant (One Lambda, Canoga Park, CA, USA). This technology has recently been implemented on the Luminex platform and has the potential for DNA-based HLA typing [27], as well as testing for antibodies to specific HLA antigens [54].
Autoimmune testing The first microsphere-based multiplexed immunoassay application that has received FDA approval is a kit for autoimmune disease testing (Zeus Scientific, Inc., Raritan, NJ, USA). IgG antibodies to the markers SSA, SSB, Jo-1, histone, Sm, RNP, Centromere B, and Scl-70 are included. Protein phosphorylation assays Traditional methods for measuring phosphorylated proteins include Western blot analysis with immunologic or isotopic identification of the modified proteins. Both procedures can involve several days of sample processing. Microparticlebased capture and detection of phosphorylated proteins is a rapid, quantifiable flow cytometric procedure [56]. Phosphorylation of the signaling protein ERK-2 has been identified with antibodies specific for the phosphorylated protein (Bio-Rad Laboratories, Hercules, CA, USA) [56]. Another method measures serine or threonine kinase activity with anti-phosphoserine and -phosphothreonine antibodies (Upstate Biotechnology).
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Single nucleotide polymorphism genotyping Single nucleotide polymorphisms (SNPs) are single-base variations found within genomic DNA [57–59]. SNPs are the most prevalent form of genetic variation. The frequency of occurrence in the human genome is approximately one SNP per every thousand bases, and it is estimated that there are about three million per genome. As the mapping of SNP loci becomes more complete, the opportunity to understand the relationship of these point mutations to disease will grow [60–61]. SNP genotyping requires methods that are accurate, high throughput, and cost effective. Typical SNP genotyping technologies included solid supports (gels, chips, glass slides), as well as mobile supports, such as mass spectrometry and capillary electrophoresis [62–64]. Recently, flow cytometric analysis of fluorescent microspheres has been identified as a novel technology platform for SNP genotype determination. Its advantages include rapid data acquisition (up to thousands of data points per second), excellent sensitivity, and multiparametric fluorescence analysis that permits multiplexed analysis. One approach to microsphere-based SNP genotyping is enzymatic allele discrimination by oligoligation assay (OLA) [65] or single base chain extension (SBCE) [66–69]. In these methods, a fluorescent reporter molecule is enzymatically added to a capture probe to determine the SNP genotype (Fig. 2). The three steps in the genotyping process are 1) allele discrimination by thermal cycling of PCR-amplified genomic DNA in a solution-based enzymatic reaction, 2) capture of the enzymatic reaction products by hybridization of DNA sequences (ZipCodes) on the capture probe with complementary sequences on the surface of the microsphere, and 3) flow cytometric analysis of the microspheres to identify both the microsphere population and the fluorescent signal associated with a genotype. The genotyping reaction components may be multiplexed for readout of multiple SNPs in a single reaction volume. For a given SNP, the OLA method allows analysis of different alleles in the same assay volume, but until systems evolve to analyze multiple fluorescent reporters, SBCE requires a separate reaction for each allele. Allele-specific primer extension (ASPE) [68,69] is another DNA polymerase-based assay. ASPE capture probes, designed as allelespecific oligos terminating in the polymorphic base, are identical to OLA capture probes. In this approach, labeled dNTPs are used instead of chain-terminating ddNTPs, allowing multiplexed SNP analysis with mixtures of allelic variants. The ZipCode concept permits use of a defined set of optimally cZipCode-coupled microspheres for different uses of SNPs. The same ZipCode sequence can be incorporated into the design of new capture probes as new SNPs are added to the assay. Other approaches to SNP genotyping have included direct or competitive hybridization of PCRamplified products to complementary sequences coupled to
Figure 2. Microsphere-based SNP genotyping by OLA and SBCE. (A) In the OLA method, the following components are thermally cycled in the presence of DNA ligase: 1) PCR-amplified DNA, encompassing the SNP to be genotyped, 2) a capture oligonucleotide probe that hybridizes to the PCRamplified DNA (including the polymorphic base) and also to a separate DNA tag sequence that has been coupled to the surface of a microsphere population (cZipCode), and 3) a short fluorescent reporter oligonucleotide that is complementary to the target DNA just after the polymorphic base. If there is base pairing between the polymorphic base on the PCR product and the capture probe, the DNA ligase will covalently couple the fluorescent reporter oligonucleotide to the capture probe. If there is no base pairing, the fluorescent reporter oligonucleotide will not be coupled. (B) In the SBCE method the following components are thermally cycled in the presence of DNA polymerase after treatment with shrimp alkaline phosphatase and Exo nuclease I: 1) PCR-amplified DNA, encompassing the SNP to be genotyped, 2) a capture oligonucleotide probe that hybridizes to the PCR-amplified DNA (its complementary 3 end stops 1 base before the polymorphic base) and also to a cZipCode, and 3) a cocktail of 4 dideoxy NTPs, only one of which is fluorescently labeled. The DNA polymerase extends the capture probe by one base. In contrast to OLA, separate reactions using different labeled ddNTPs must be performed for each SNP allele; i.e., a biallelic SNP requires two reactions. In each method, the enzymatic reaction products are hybridized to the surface of the fluorescent microspheres by the ZipCode sequences on the capture probe. Flow cytometric analysis of the microspheres identifies both the microsphere population and the fluorescent signal associated with a genotype.
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microspheres [70,71]. These approaches require a unique oligonucleotide-coupled microsphere population to match each SNP allele and can require an elevated temperature during the flow cytometric analysis. The advantages of microsphere-based SNP genotyping include greater flexibility than static platforms such as the chip, excellent accuracy at a reasonable cost, and increased throughput by multiplexing. A limiting factor for high throughput is the technical difficulty in generating large numbers of individual PCR samples. Incorporating multiplexed PCR promises to yield even greater throughput. As with any high-throughput method that generates data, the challenges include integrating a robust information-management system to handle the deluge of data and extract reliable information. In addition to SNP genotyping, microsphere-based DNA techniques have been applied to the identification of bacterial [69,72,73] and viral samples [74–77]. While most have used hybridization of PCR-amplified samples to complementary sequence-coupled microspheres, Ye et al. [69] have used ASPE and SBCE to generate bacterial species- and genusspecific binding patterns with 16S rDNA. Microsphere technology has also been applied to the detection of translocationassociated fusion genes associated with leukemias [78] and screening for gene mutations such as the cystic fibrosis transmembrane conductance regulator gene [79] and -globin gene [80], as well as profiling gene expression [81–83]. Collectively, these approaches promise to expand the applications of flow cytometry well beyond cell-based analysis. Nuclear receptor binding Nuclear receptors are ligand-dependent transcription factors that regulate genes involved in a wide variety of biological processes such as cell development and metabolism [84– 86]. One mechanism by which nuclear receptors achieve their regulatory function is ligand-modulated binding to a large group of proteins called coactivator and corepressor proteins [87–89]. These interactions take place in a region of the nuclear receptor protein called the ligand binding domain (LBD). Coactivator proteins bind to the nuclear receptor LBD through signature LXXLL binding motifs, where L represents leucine and X can represent any amino acid [90]. The LXXLL sequence has been shown to form a two-turn amphipathic helix where the aliphatic side chains of the leucine residues are spatially oriented to interact with a key hydrophobic region in the nuclear receptor LBD. Coactivator proteins have been reported to possess intrinsic histone acetyl transferase activity [91], which is thought to facilitate transcription by relaxing chromatin structure. Coactivator proteins are quite large and may contain several LXXLL motifs; for example, the coactivator protein steroid receptor coactivator-1 (SRC-1) (MW 114 KD) contains four LXXLL motifs. Short synthetic peptides containing LXXLL sequences have been used as tools to explore coactivator protein–nuclear receptor interactions [90,92,93].
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X-ray crystallography of nuclear receptors has demonstrated that small molecule ligands bind within a hydrophobic pocket of the LBD and that this can impact nuclear receptor protein conformation [94]. In studies of the estrogen receptor (ER), the ligands diethystilbesterol, estrogen, and genistein were found to stabilize a nuclear receptor conformation that promotes coactivator binding along a hydrophobic cleft on the receptor surface. In contrast, the ligands tamoxifen or raloxifene do not promote coactivator binding. In fact, these ligands stabilize a protein conformation where the AF-2 helix of the LBD physically occludes the coactivator binding cleft [95–97]. Most likely, in the absence of ligand, different conformational states exist in thermodynamic equilibrium. Upon ligand binding, the proportion of these states is altered. By designing small molecules that have selective effects on protein-protein interactions, and by efficiently evaluating these interactions, there are opportunities to discover new transcriptional modulators with therapeutic benefit. Microsphere technology has been applied to the study of molecular interactions of nuclear receptors. In addition to saving time and reagents, the microsphere approach has extended traditional binding methodologies, such as timeresolved fluorescence resonance energy transfer and surface plasmon resonance, by evaluating compound effects on interactions in the context of many other interactions. Using microspheres, different peptide sequences that represent the binding motifs of coactivator proteins are coupled to the surface of fluorescent microsphere populations (Fig. 3). Fluorochrome-labeled purified recombinant nuclear receptor LBD is added to the multiplexed microsphere mix and incubated in the absence or presence of ligand. The binding of the nuclear receptor to the peptide sequences and decoding of the microsphere populations is done by flow cytometry in a no-wash assay. Microspheres have been used to demonstrate quantitative binding differences between the nuclear receptors ER LBD and PPAR LBD and coactivator peptides, as well as to determine the effect of small molecule ligands on these binding interactions [98]. The ligand estradiol enhanced ER and ER LBD binding to microsphere-coupled coactivator peptides while the antiestrogenic ligands tamoxifen or raloxifene inhibited binding [98,99]. When evaluating quantitative binding differences using microsphere technology, the density of the molecule coupled to the microsphere surface can have a direct influence on receptor binding affinity [98]. This is demonstrated by an experiment where coactivator peptide was coupled to microspheres at different densities (5,000 to 1,000,000 peptide molecules per microsphere). As the peptide density increased to 200,000 molecules per microsphere, the apparent affinity of receptor binding also increased (i.e., KD decreased). However, at densities higher than 200,000 molecules per microsphere, the effect on KD was negligible. Most likely, matrix or lattice-like effects on avidity produce these effects. The
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Figure 3. Molecular interactions of nuclear receptors. Microsphere populations coupled to peptide sequences that represent the binding motifs of coactivator proteins are incubated with fluorochrome-labeled nuclear receptor LBD in the absence or presence of small molecule ligand. The binding of the nuclear receptor to the peptide sequences and decoding of the microsphere populations is determined by flow cytometry in a no-wash assay.
close proximity of neighboring binding partners increases the likelihood that a receptor will rebind another peptide rather than becoming free in solution by dissociation. Highdensity conditions reduce the apparent off-rate and result in higher apparent affinities. When comparing binding affinities between different coupled molecules, coupling density should be at a level where avidity rebinding is no longer effected by density. In contrast, one can take advantage of site density to optimize a multiplexed assay to look at partners that vary in affinity. For example, in experiments that require the binding signal of all interacting molecules to be equivalent for a given receptor concentration, a low-affinityinteracting molecule may be coupled at high density and high-affinity-interacting molecules at low density. Nuclear receptor regulation of gene expression involves a host of complex binding interactions. In addition to homoor heterodimerization, nuclear receptors interact with coactivator proteins, corepressor proteins, and DNA. Microsphere technology can play an important role in understanding these complex molecular mechanisms and, in principle, may be applied to any array of interacting molecules. The future for microsphere arrays The future for microsphere assays is unlimited. Clinical diagnostics can be revolutionized by multiplexed microsphere technology that reduces the cost per test and turnaround times for diagnoses. A number of potential research applications and the increased speed of drug discovery and testing are just beginning to be explored. Microsphere technology will be improved by the availability of high-affinity antibodies and instrument calibration particles. With the current instrumentation and available antibodies, the limit of detection for cytokines, in particular, is in the low picogram and high femtogram range. The challenge is to push this limit of sensitivity for analytes that are relevant
to clinical and research applications consistently into the femtogram range. In addition, there is also a need for quality control particles that monitor fluorescence detector linearity and allow the conversion of fluorescence intensity values to molecules of equivalent soluble fluorochrome (MESF) [100]. Although these products are manufactured for conventional flow cytometers, they are not adaptable for the microspherededicated Luminex instrument. A significant advantage to multiplexing is that internal controls for the addition of the correct sample volume, detection of inhibitory immunoglobulins, nonspecific binding, and instrument performance can be monitored by including microspheres with the relevant properties [9,30,68]. Additional controls, such as those for MESF determination, could also be added to permit automated quantitation and quality control with each sample. It is anticipated that a number of companies will offer their own versions of a miniaturized flow cytometer for analysis of microsphere arrays. A high-throughput instrument that processes 600 to 1000 samples per hour has been released recently (HTS, Luminex Corp.). A fully automated instrument complete with reagent dispensing and microplate handling would maximize the potential of this technology. Undoubtedly, the microsphere chemistries will be improved to expand the capture, binding, and detection capabilities. An array that follows three or more dyes per microsphere that follows the Luminex strategy of 10 intensities of each dye (10n) would expand the size of an array into the thousands. A new spectral coding system that uses semiconductor quantum-dots (QDs), such as ZnS-capped CdSe nanocrystals, embedded into polymer beads has been described [101,102]. These QDs have narrow emission peaks and the realistic potential for 10,000 to 40,000 recognizable codes. Since most high-throughput screening assays are based on ligand coupling and/or a solid support matrix, many assay formats can be supported by fluorescence microspherebased detection. The available dyes and microsphere compositions can accommodate a variety of arrays. Microsphere-based methods should provide a rapid alternative to the methods currently used for drug susceptibility testing, phage display screening, gene expression analysis, etc., as well as the other applications discussed in the text. Once several hundred rather than 100,000 genes of interest are identified by DNA chip- or slide-based microarrays, microsphere arrays will provide a less expensive and more easily modified platform for further study of specific gene subsets. Microsphere technology holds great promise as a tool to probe both genomic and proteomic function. Acknowledgments We appreciate the assistance of J. David Taylor, Dr. Kenneth H. Pearce, and Dr. Kerry Oliver for their critical reading of the manuscript and Dr. Abbas Vafai and Dr. John G. Gray for their support of microsphere-based assay technology at the Centers for Disease Control and Prevention and GlaxoSmithKline, respectively.
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Multiplexed microsphere-based flow cytometric assays Kathryn L. Kellara and Marie A. Iannoneb a National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Ga., USA; bGlaxoSmithKline, Research Triangle Park, NC, USA
Flow cytometry has become an indispensable tool for clinical diagnostics and basic research. Although primarily designed for cellular analysis, flow cytometers can detect any particles in the lower micron range, including inert microspheres of different sizes, dyed with various fluorochromes. Over the past 20 years, microspheres have been used as calibrators for flow cytometers and also as a solid support for numerous molecular reactions quantitated by flow cytometry. Proteins, oligonucleotides, polysaccharides, lipids, or small peptides have been adsorbed or chemically coupled to the surface of microspheres to capture analytes that are subsequently measured by a fluorochrome-conjugated detection molecule. More recently, assays for similar analytes have been multiplexed, or analyzed in the same assay volume, by performing each reaction on a set of microspheres that are dyed to different fluorescent intensities and, therefore, are spectrally distinct. Some recent applications with fluorescent microspheres have included cytokine quantitation, single nucleotide polymorphism genotyping, phosphorylated protein detection, and characterization of the molecular interactions of nuclear receptors. The speed, sensitivity, and accuracy of flow cytometric detection of multiple binding events measured in the same small volume have the potential to replace many clinical diagnostic and research methods and deliver data on hundreds of analytes simultaneously. © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.
Flow cytometry has been used for the multiparametric analysis of hematologic and immunologic cells for over 20 years. Although the majority of the applications are cell based, particles such as microspheres can also be detected by laser light scatter. Microspheres have a considerable history as the solid support for a variety of molecular reactions that are analyzed by flow cytometry. As early as 1977, flow cytometry was applied to the detection of antigens captured by antibodies coated on the surface of microspheres [1]. In addition, polymerase chain reaction (PCR) products have been detected by hybridization to oligonucleotide probes bound to microspheres and enzymatic reaction products have been captured on microspheres [2,3]. These basic molecular applications have been expanded to the simultaneous detection of multiple analytes in the same sample volume by combining individual reactions on spectrally distinct sets of microspheres. This review will explore the expansion of flow cytometry to the detection of these multiplexed molecular reactions on an array of fluorescent microspheres.
Offprint requests to: Kathryn L. Kellar, Ph.D., CDC/NCID/SRP, MS D-34, 1600 Clifton Rd., NE, Atlanta, GA 30333 USA; E-mail: klk@ cdc.gov
Microsphere technology Historical perspective Antibodies and antigens have been adsorbed or directly or indirectly coupled to microspheres composed of a variety of materials, such as latex, polystyrene, polyacrylamide, and glass [4–7]. Immunoassays based on the physical adsorption of antibodies to microspheres for analyte quantitation include ones for -2 microglobulin [8], Clostridium difficile toxin [5], and -fetoprotein [9]. Human IgG [6] and IgE [10] have been measured with antibodies chemically coupled to microspheres. Likewise, antigens have been adsorbed to microspheres to detect antibodies specific for Helicobacter pylori [11], human IgA [12], light chains [13], and phospholipids [14–16] or covalently attached to detect antibodies to -gliadin [17]. Microspheres coated with human C1q were used to detect circulating immune complexes containing elusive HIV antigens [18,19] and collagen has been used to capture vWF [7]. Although flow cytometric detection of PCR and enzymatic reaction products captured on microspheres was not widely employed until recently, there were a few early reports. The activity of potential inhibitors of gelatinase B was monitored by the fluorescence of substrates adsorbed to microspheres [3]. Flow cytometric detection of fluorescein-
0301-472X/02 $–see front matter. Copyright © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(02)0 0 9 2 2 - 0
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ated PCR products captured on oligonucleotide- or streptavidin-coated magnetic beads was shown to be more sensitive and less hazardous than traditional methods, such as dye- or radioisotope-resolved gel electrophoresis or chemiluminescence of filter-hybridized products [2,20,21]. The unique properties of microspheres, which include their range of sizes, stability, uniformity, and capacity to absorb and retain fluorescent dye, were readily adapted to flow cytometric analysis of multiplexed reactions. Some of the first multiplexed immunoassays detected antibodies to cytomegalovirus and herpes simplex virus [22], different Candida albicans preparations [23], hepatitis C core and nonstructural proteins [24], and four recombinant DNA-produced HIV proteins [25] after capture on antigen-coated microspheres of different sizes.
charides can be purchased. Noncovalent linkages are useful for some applications. Species-specific anti-IgGs coupled to microspheres facilitate direct binding of immunoglobulins from ascites fluids or concentrated hybridoma supernatants when further purification is not practical. High- and lowdensity streptavidin-coated fluorospheres with the capacity to bind biotinylated analytes are available for adjusting the dynamic range of quantitative assays and signal-to-noise ratios (Radix Biosolutions, Georgetown, TX, USA). Similarly, other affinity tags such as glutathione-GST, Ni-6xhistidine, and protein A and G can be utilized to link capture proteins to microspheres (Upstate Biotechnology, Waltham, MA, USA; QIAGEN Sciences, Valencia, CA, USA). Procedures for covalent and noncovalent protein coupling to microspheres have been reviewed previously [26].
Microsphere availability Microspheres of various sizes and composed of different polymers are available from a number of vendors (Table 1). Several companies offer an “array” of fluorescent microspheres with different emission spectra and intensities that permit the simultaneous analysis of several to potentially a hundred analytes. Fluorescent microspheres with carboxyl, amine/hydrazide, and maleimide groups for covalent coupling of proteins, peptides, oligonucleotides, and polysac-
Instrumentation Conventional flow cytometers with the appropriate lasers for the respective fluorochromes can acquire microspherebased emission data. In the mid-1990s, the Luminex Corporation (Austin, TX, USA) coupled a digital signal processor and a Becton-Dickinson benchtop cytometer equipped with a 488-nm argon laser to detect and process thousands of fluorescent signals generated by multiplexed microspherebased assays in real time (FlowMetrix System) [27]. With
Table 1. Resources for multiplexed microsphere-based flow cytometric assays* Source†
Web site
Instrumentation, software, and kits
Applied Cytometry Systems BD Biosciences Bio-Rad Laboratories MiraiBio, Inc. Luminex Corp.
appliedcytometry.biz bdbiosciences.com/pharmingen bio-rad.com miraibio.com luminexcorp.com
Microspheres
Bangs Labs Duke Scientific Luminex Corp. Molecular Probes Polysciences, Inc. Qiagen Radix BioSolutions Seradyn Spherotech Upstate Biotechnology
bangslabs.com dukescientific.com luminexcorp.com molecularprobes.com polysciences.com qiagen.com radixbiosolutions.com seradyn.com spherotech.com upstatebiotech.com
Multiplexed immunoassay kits
Bender MedSystems BioErgonomics Biosource International LINCO Research, Inc. One Lambda R & D Systems Upstate Biotechnology Zeus Scientific, Inc.
bendermedsystems.com bioe.com biosource.com lincoresearch.com onelambda.com rndsystems.com upstatebiotech.com zeusscientific.com
Software
Brendan Scientific
brendan.com
Resource
*This list includes some of the companies that have products for multiplexed fluorescent microsphere-based assays † The use of trade names and commercial sources is for identification purposes only and does not imply endorsement by the Centers for Disease Control and Prevention or the Department of Health and Human Services.
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this system, 64 sets of microspheres dyed with different intensities of two fluorophores were available for multiplexed assay development [28]. A disadvantage of the FlowMetrix system was the need to compensate for the overlap in fluorescence between the reporter (FL1 fluorescence) and the microspheres (FL2/FL3 fluorescence). This system was replaced with an instrument specifically designed to analyze fluorescent microsphere-based assays. The Luminex 100 and proprietary microspheres were developed to provide a high signal-to-noise ratio for detection of low-level fluorescence with PE as the reporter (Luminex Corp.). The Luminex 100 is a compact benchtop cytometer equipped with a digital signal processor and two inexpensive lasers. The 532-nm, 13-mW yttrium aluminum garnet (YAG) laser was selected for improved excitation of R-phycoerythrin (PE, 578-nm emission) compared with a 488-nm laser. The 635-nm 10-mW red diode laser excites the two classification fluorochromes embedded within the proprietary 5.6-m microspheres. Different ratios of these dyes define 100-microsphere populations. The dyes emit at 658 and 712 nm and have no spectral overlap with reporters such as PE or Alexa 532, so no fluorescent compensation is required [29,30]. An optional X-Y platform for reading 96-well microtiter plates facilitates automated sample acquisition. With this platform, both hardbottom and filter-bottom “wash” plates (Millipore Multiscreen, Burlington, MA, USA) can be processed.
Multiplexed microsphere applications Immunoassays Multiple immunoassays can be performed simultaneously on an array of microspheres as long as the conditions of the assays (incubation times, sequence of reagent additions, washes) are the same or can be combined. For quantitative sandwich immunoassays, standard curves are optimized one analyte at a time, with the capture antibody coupled to the microsphere and the detection antibody bound to the reporter (Fig. 1). The general principles of ELISA development, which are to titer each reagent and modify incubation times, temperatures, and washing conditions, are applicable. Often microsphere-based assays can be performed with no wash steps if the background and potential interfering substances in the samples are minimal. They can also be done in one step [31,32]. Since each microsphere population is distinguished by size and/or spectral address, the reporter intensity relates to the quantity of analyte bound and thus establishes the relative concentration of that analyte. Commercial antibody pairs optimal for ELISA development may not always be optimal for multiplexed microsphere immunoassays. Antibodies should be tested to find the best pairs, be matched for affinity, and not be reactive with the same epitope [33]. The most stringent assay conditions for the weakest pair of antibodies define the conditions of the multiplexed assay. Once optimized, the individual as-
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says can be combined into the reaction volumes of one assay. The cross-reactivity of all analytes and antibodies in a multiplexed format must be minimal. A primary difficulty encountered with multiplexed immunoassays is the false-positive or -negative results from heterophilic antibodies, human anti-animal antibodies, rheumatoid factor (RF), soluble receptors for the analyte, etc., similar to those observed with human serum or plasma samples in two-site assays such as ELISAs [34–39]. Multiple antibody pairs in a multiplexed format compound the potential for interference. For these assays to be effective, the standards and blanks should be diluted in a matrix similar to the samples, i.e., cell culture supernatants, serum, etc., and blocking agents similar to those used for ELISAs should be added [40]. Fixation with a final 1% formaldehyde or similar fixative is often necessary to ensure that reaction times are the same for standards and samples that may be read over the span of 30 to 60 minutes on a cytometer [40]. Fixation also inactivates any potential biohazards in human body fluids that fall under the guidelines of the OSHA Regulations for Bloodborne Pathogens (osha.gov/OSHStd_data/1910_1030. html). Unfixed human samples should not be read directly on instruments in the open laboratory. The steps from setup to data analysis of a microspherebased immunoasssay are outlined in Figure 1. An antibody detection and a sandwich immunoassay format are diagrammed, but any immunoassay may be designed. The fluorescent intensities of each fluorochrome are measured and, if quantitative, the assay data are transformed with analytical software. In this example, the microsphere populations are classified with two fluorochromes and a third fluorescent reporter, PE, is used to quantitate eight cytokine concentrations. The 8 data points for each sample are derived from a minimum of 2400 signals (8 populations of microspheres, 100 microspheres acquired per population, 3 fluorescent signals per microsphere). Although the speed of multiplexed assay performance and data acquisition is a definite advantage, the speed of data analysis is just as critical for high-throughput data processing. If an array of 100 sets of microspheres is set up in each of 96 wells, a minimum of 2,880,000 signals need to be condensed into a manageable data output. Several analytical software packages that perform statistical curve fitting and compute analyte concentrations in real time are currently available (Table 1) [32,41]. Quantitation of cytokines Cytokines, chemokines, and growth factors (hereafter referred to collectively as cytokines) are excellent candidates for multiplexed analysis because of their complex interrelationships in immunologic and hematologic functions. Fluctuations in the level of one cytokine often induce changes in others. Thus, the rates of secretion of these analytes in a variety of physiological conditions are best measured simultaneously in identical reaction formats.
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Figure 1. Steps in a multiplexed microsphere-based flow cytometric immunoassay. (A) Antibody detection and sandwich immunoassay formats performed on microspheres in liquid suspension are diagrammed. Any antigen-antibody-based format can be designed with a terminal reporter molecule specific to the laser detection system and distinct from any fluorescence of the microspheres. (B) At the completion of the binding assay, fluorescent intensities associated with each microsphere are measured by flow cytometry. In this example, the optical detection system of the Luminex 100 is illustrated (see text). (C) The dot plot displays 100 distinct regions for each Luminex microsphere population classified on the basis of the intensities of two internal fluorochromes. The instrument was set to acquire at least 100 microspheres each of 8 spectrally distinct populations of microspheres. (D) Multiplexed data from a random sample in an 8-plex assay was calculated from the log-log transformation of the values for the standards using SOFTmax PRO software (Molecular Devices, Sunnyvale, CA, USA) and graphed with Excel 2000 software (Microsoft Corp., Redmond, WA, USA).
Multiplexed microsphere arrays for cytokines developed by commercial vendors or independent laboratories are primarily two-site immunoassays. Relatively high-affinity matched pairs of antibodies for ELISAs for at least 50 human cytokines and related molecules are available from commercial vendors. The limit of sensitivity of these commercial antibody pairs falls in the low picogram range (R & D Systems, Inc., Minneapolis, MN, USA; Pharmingen/BD Biosciences, San Jose, CA, USA; among others). This limit derives from high-affinity antibodies (KD 108 to 1010) that, in combination with liquid-phase kinetics and the high binding capacity of three-dimensional microspheres, produce a robust multiplexed immunoassay. The full range of these assays extends over 3 to 4 logs, as compared with 1 to 2 logs for ELISAs. A multiplexed array for 15 mouse cytokines is the largest described to date [33]. The FlowMetrix system was employed to study the secretion of TH1 vs TH2 cytokines from
stimulated mouse CD4 T cells. Overall patterns of cytokine secretion were similar with either microsphere or ELISA methods. However, the FlowMetrix assay had a lower limit of sensitivity because a reduction in the number of microspheres reacted per cytokine and extension of the incubation times increased the reporter signals. The multiplexed assay was also more reproducible. The data were derived from measurements of at least 100 microspheres per cytokine, with each microsphere functioning as an independent assay. These measurements were more reliable than a single reading from the bottom of a microtiter well for ELISAs [33]. Multiplexed assays for up to nine human cytokines have been described [31,32,40–43]. Different combinations of interleukins (IL)-1, -2, -4, -5, -6, -8, -10, -12, TNF-, IFN-, GM-CSF, IFN-inducible protein (IP)-10, and RANTES were measured in whole blood, cell-culture supernatants, tears, and serum. The performance of the multiplexed formats for human cytokines has been validated by compari-
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sons with ELISAs [31,32,40–42]. Based on the historical variability of results obtained with different ELISA kits for the same antigen, it is fair to say that comparisons between multiplexed assays and ELISAs are most valid if the same antibodies and cytokine standards are used in both formats. Such comparisons demonstrated that the detection limits were the same or better for the multiplexed assays [31, 32,40–42]. Sample data from the two methods were highly comparable (correlation coefficients 0.857 to 1.0) [31,32,40,42]. Reproducibility was excellent for the multiplexed format: intraassay CVs were less than 10% [31,41] and interassay CVs were less than 5% [41]. Recoveries of 8 cytokines spiked into a buffer or normal serum matrix averaged 102.5 5% (n 13) [40]. Microsphere-based arrays are more rapid, less expensive, and require less sample volume than ELISAs [32,33,40– 42,44]. In the near-liquid phase of the former, equilibrium is reached sooner so incubation times are reduced significantly, particularly if wash steps are eliminated. Based on an assay for 8 cytokines (8-plex), Kellar and colleagues calculated that 8 ELISA kits cost 17 times that of multiplex reagents (Kellar, unpublished observations) and the 8-plex required less than or equal to 1/20th the amount of sample (20 L vs 400 L for ELISAs) [40]. Other methods for measuring cytokine production are ELISPOT and intracellular staining [45,46]. Neither technique is quantitative; both measure the frequency of cells producing detectable levels of specific cytokines. The ELISPOT technique detects the secretion of a specific cytokine from individual cells deposited in the wells of a multi-well plate. The wells are surface-coated with antibody and subsequently developed for a colorimetric reaction, similarly to ELISAs. The number of cytokine-producing cells is more often manually enumerated [45]. Intracellular cytokine detection by flow cytometry also measures cytokine production by single cells. Intracellular detection is accomplished by fixation and permeabilization of cells followed by specific antibody staining. With this technique, multiple cytokines as well as cell types can be identified by multiparametric analysis of a heterogeneous cell population [46]. By combining various techniques such as ELISPOT or intracellular staining with microsphere-based assays to quantitate secreted cytokine and methods that measure cytokine receptors, a comprehensive analysis of the regulation of cytokine production can be made [47].
Analytes in newborn blood spots A microsphere-based test for congenital hypothyroidism has been reported [48]. Proteins eluted from dried blood spots were analyzed by combining two microsphere assays, one a competitive assay for thyroxine (T4) and the other a sandwich immunoassay for thyrotropin (TSH). Similarly, HIV-1 antigens on microspheres were used to detect HIV antibodies [49].
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Viral and bacterial identification Two groups have reported bacterial serotyping of multiple strains of Streptococcus pneumoniae [50,51]. Both assays were based on microspheres coated with strain-specific polysaccharides and binding of serum IgG antibodies, followed by a fluorescent secondary antibody. A measure of viral load was developed by covalently coupling adenovirus antibodies to microspheres and incubating with DNAspecific fluorophores [52]. The mean fluorescence intensity was measured from a standard curve of fluorescent intensity vs viral concentration. The identification of bacteria and viruses by DNA-based methods is described in the single nucleotide polymorphism genotyping section of this review. Fluorosphere-based analyses would be applicable for disease surveillance [53], vaccine development and surveillance, screening of hybridomas and donated blood, and other highthroughput screening procedures. Jani et al. [53] have proposed that multiplexed immunoassays for sexually transmitted diseases, childhood diseases presenting with rash and fever, or testing critical to blood banking could be performed on a small, inexpensive flow cytometer placed in the underserved regions of the world for healthcare delivery and disease surveillance.
HLA testing Flow cytometry has significantly impacted HLA testing [54,55]. Microspheres coated with HLA class I and class II antigens have been used to test for HLA antibodies prior to transplant (One Lambda, Canoga Park, CA, USA). This technology has recently been implemented on the Luminex platform and has the potential for DNA-based HLA typing [27], as well as testing for antibodies to specific HLA antigens [54].
Autoimmune testing The first microsphere-based multiplexed immunoassay application that has received FDA approval is a kit for autoimmune disease testing (Zeus Scientific, Inc., Raritan, NJ, USA). IgG antibodies to the markers SSA, SSB, Jo-1, histone, Sm, RNP, Centromere B, and Scl-70 are included. Protein phosphorylation assays Traditional methods for measuring phosphorylated proteins include Western blot analysis with immunologic or isotopic identification of the modified proteins. Both procedures can involve several days of sample processing. Microparticlebased capture and detection of phosphorylated proteins is a rapid, quantifiable flow cytometric procedure [56]. Phosphorylation of the signaling protein ERK-2 has been identified with antibodies specific for the phosphorylated protein (Bio-Rad Laboratories, Hercules, CA, USA) [56]. Another method measures serine or threonine kinase activity with anti-phosphoserine and -phosphothreonine antibodies (Upstate Biotechnology).
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Single nucleotide polymorphism genotyping Single nucleotide polymorphisms (SNPs) are single-base variations found within genomic DNA [57–59]. SNPs are the most prevalent form of genetic variation. The frequency of occurrence in the human genome is approximately one SNP per every thousand bases, and it is estimated that there are about three million per genome. As the mapping of SNP loci becomes more complete, the opportunity to understand the relationship of these point mutations to disease will grow [60–61]. SNP genotyping requires methods that are accurate, high throughput, and cost effective. Typical SNP genotyping technologies included solid supports (gels, chips, glass slides), as well as mobile supports, such as mass spectrometry and capillary electrophoresis [62–64]. Recently, flow cytometric analysis of fluorescent microspheres has been identified as a novel technology platform for SNP genotype determination. Its advantages include rapid data acquisition (up to thousands of data points per second), excellent sensitivity, and multiparametric fluorescence analysis that permits multiplexed analysis. One approach to microsphere-based SNP genotyping is enzymatic allele discrimination by oligoligation assay (OLA) [65] or single base chain extension (SBCE) [66–69]. In these methods, a fluorescent reporter molecule is enzymatically added to a capture probe to determine the SNP genotype (Fig. 2). The three steps in the genotyping process are 1) allele discrimination by thermal cycling of PCR-amplified genomic DNA in a solution-based enzymatic reaction, 2) capture of the enzymatic reaction products by hybridization of DNA sequences (ZipCodes) on the capture probe with complementary sequences on the surface of the microsphere, and 3) flow cytometric analysis of the microspheres to identify both the microsphere population and the fluorescent signal associated with a genotype. The genotyping reaction components may be multiplexed for readout of multiple SNPs in a single reaction volume. For a given SNP, the OLA method allows analysis of different alleles in the same assay volume, but until systems evolve to analyze multiple fluorescent reporters, SBCE requires a separate reaction for each allele. Allele-specific primer extension (ASPE) [68,69] is another DNA polymerase-based assay. ASPE capture probes, designed as allelespecific oligos terminating in the polymorphic base, are identical to OLA capture probes. In this approach, labeled dNTPs are used instead of chain-terminating ddNTPs, allowing multiplexed SNP analysis with mixtures of allelic variants. The ZipCode concept permits use of a defined set of optimally cZipCode-coupled microspheres for different uses of SNPs. The same ZipCode sequence can be incorporated into the design of new capture probes as new SNPs are added to the assay. Other approaches to SNP genotyping have included direct or competitive hybridization of PCRamplified products to complementary sequences coupled to
Figure 2. Microsphere-based SNP genotyping by OLA and SBCE. (A) In the OLA method, the following components are thermally cycled in the presence of DNA ligase: 1) PCR-amplified DNA, encompassing the SNP to be genotyped, 2) a capture oligonucleotide probe that hybridizes to the PCRamplified DNA (including the polymorphic base) and also to a separate DNA tag sequence that has been coupled to the surface of a microsphere population (cZipCode), and 3) a short fluorescent reporter oligonucleotide that is complementary to the target DNA just after the polymorphic base. If there is base pairing between the polymorphic base on the PCR product and the capture probe, the DNA ligase will covalently couple the fluorescent reporter oligonucleotide to the capture probe. If there is no base pairing, the fluorescent reporter oligonucleotide will not be coupled. (B) In the SBCE method the following components are thermally cycled in the presence of DNA polymerase after treatment with shrimp alkaline phosphatase and Exo nuclease I: 1) PCR-amplified DNA, encompassing the SNP to be genotyped, 2) a capture oligonucleotide probe that hybridizes to the PCR-amplified DNA (its complementary 3 end stops 1 base before the polymorphic base) and also to a cZipCode, and 3) a cocktail of 4 dideoxy NTPs, only one of which is fluorescently labeled. The DNA polymerase extends the capture probe by one base. In contrast to OLA, separate reactions using different labeled ddNTPs must be performed for each SNP allele; i.e., a biallelic SNP requires two reactions. In each method, the enzymatic reaction products are hybridized to the surface of the fluorescent microspheres by the ZipCode sequences on the capture probe. Flow cytometric analysis of the microspheres identifies both the microsphere population and the fluorescent signal associated with a genotype.
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microspheres [70,71]. These approaches require a unique oligonucleotide-coupled microsphere population to match each SNP allele and can require an elevated temperature during the flow cytometric analysis. The advantages of microsphere-based SNP genotyping include greater flexibility than static platforms such as the chip, excellent accuracy at a reasonable cost, and increased throughput by multiplexing. A limiting factor for high throughput is the technical difficulty in generating large numbers of individual PCR samples. Incorporating multiplexed PCR promises to yield even greater throughput. As with any high-throughput method that generates data, the challenges include integrating a robust information-management system to handle the deluge of data and extract reliable information. In addition to SNP genotyping, microsphere-based DNA techniques have been applied to the identification of bacterial [69,72,73] and viral samples [74–77]. While most have used hybridization of PCR-amplified samples to complementary sequence-coupled microspheres, Ye et al. [69] have used ASPE and SBCE to generate bacterial species- and genusspecific binding patterns with 16S rDNA. Microsphere technology has also been applied to the detection of translocationassociated fusion genes associated with leukemias [78] and screening for gene mutations such as the cystic fibrosis transmembrane conductance regulator gene [79] and -globin gene [80], as well as profiling gene expression [81–83]. Collectively, these approaches promise to expand the applications of flow cytometry well beyond cell-based analysis. Nuclear receptor binding Nuclear receptors are ligand-dependent transcription factors that regulate genes involved in a wide variety of biological processes such as cell development and metabolism [84– 86]. One mechanism by which nuclear receptors achieve their regulatory function is ligand-modulated binding to a large group of proteins called coactivator and corepressor proteins [87–89]. These interactions take place in a region of the nuclear receptor protein called the ligand binding domain (LBD). Coactivator proteins bind to the nuclear receptor LBD through signature LXXLL binding motifs, where L represents leucine and X can represent any amino acid [90]. The LXXLL sequence has been shown to form a two-turn amphipathic helix where the aliphatic side chains of the leucine residues are spatially oriented to interact with a key hydrophobic region in the nuclear receptor LBD. Coactivator proteins have been reported to possess intrinsic histone acetyl transferase activity [91], which is thought to facilitate transcription by relaxing chromatin structure. Coactivator proteins are quite large and may contain several LXXLL motifs; for example, the coactivator protein steroid receptor coactivator-1 (SRC-1) (MW 114 KD) contains four LXXLL motifs. Short synthetic peptides containing LXXLL sequences have been used as tools to explore coactivator protein–nuclear receptor interactions [90,92,93].
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X-ray crystallography of nuclear receptors has demonstrated that small molecule ligands bind within a hydrophobic pocket of the LBD and that this can impact nuclear receptor protein conformation [94]. In studies of the estrogen receptor (ER), the ligands diethystilbesterol, estrogen, and genistein were found to stabilize a nuclear receptor conformation that promotes coactivator binding along a hydrophobic cleft on the receptor surface. In contrast, the ligands tamoxifen or raloxifene do not promote coactivator binding. In fact, these ligands stabilize a protein conformation where the AF-2 helix of the LBD physically occludes the coactivator binding cleft [95–97]. Most likely, in the absence of ligand, different conformational states exist in thermodynamic equilibrium. Upon ligand binding, the proportion of these states is altered. By designing small molecules that have selective effects on protein-protein interactions, and by efficiently evaluating these interactions, there are opportunities to discover new transcriptional modulators with therapeutic benefit. Microsphere technology has been applied to the study of molecular interactions of nuclear receptors. In addition to saving time and reagents, the microsphere approach has extended traditional binding methodologies, such as timeresolved fluorescence resonance energy transfer and surface plasmon resonance, by evaluating compound effects on interactions in the context of many other interactions. Using microspheres, different peptide sequences that represent the binding motifs of coactivator proteins are coupled to the surface of fluorescent microsphere populations (Fig. 3). Fluorochrome-labeled purified recombinant nuclear receptor LBD is added to the multiplexed microsphere mix and incubated in the absence or presence of ligand. The binding of the nuclear receptor to the peptide sequences and decoding of the microsphere populations is done by flow cytometry in a no-wash assay. Microspheres have been used to demonstrate quantitative binding differences between the nuclear receptors ER LBD and PPAR LBD and coactivator peptides, as well as to determine the effect of small molecule ligands on these binding interactions [98]. The ligand estradiol enhanced ER and ER LBD binding to microsphere-coupled coactivator peptides while the antiestrogenic ligands tamoxifen or raloxifene inhibited binding [98,99]. When evaluating quantitative binding differences using microsphere technology, the density of the molecule coupled to the microsphere surface can have a direct influence on receptor binding affinity [98]. This is demonstrated by an experiment where coactivator peptide was coupled to microspheres at different densities (5,000 to 1,000,000 peptide molecules per microsphere). As the peptide density increased to 200,000 molecules per microsphere, the apparent affinity of receptor binding also increased (i.e., KD decreased). However, at densities higher than 200,000 molecules per microsphere, the effect on KD was negligible. Most likely, matrix or lattice-like effects on avidity produce these effects. The
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Figure 3. Molecular interactions of nuclear receptors. Microsphere populations coupled to peptide sequences that represent the binding motifs of coactivator proteins are incubated with fluorochrome-labeled nuclear receptor LBD in the absence or presence of small molecule ligand. The binding of the nuclear receptor to the peptide sequences and decoding of the microsphere populations is determined by flow cytometry in a no-wash assay.
close proximity of neighboring binding partners increases the likelihood that a receptor will rebind another peptide rather than becoming free in solution by dissociation. Highdensity conditions reduce the apparent off-rate and result in higher apparent affinities. When comparing binding affinities between different coupled molecules, coupling density should be at a level where avidity rebinding is no longer effected by density. In contrast, one can take advantage of site density to optimize a multiplexed assay to look at partners that vary in affinity. For example, in experiments that require the binding signal of all interacting molecules to be equivalent for a given receptor concentration, a low-affinityinteracting molecule may be coupled at high density and high-affinity-interacting molecules at low density. Nuclear receptor regulation of gene expression involves a host of complex binding interactions. In addition to homoor heterodimerization, nuclear receptors interact with coactivator proteins, corepressor proteins, and DNA. Microsphere technology can play an important role in understanding these complex molecular mechanisms and, in principle, may be applied to any array of interacting molecules. The future for microsphere arrays The future for microsphere assays is unlimited. Clinical diagnostics can be revolutionized by multiplexed microsphere technology that reduces the cost per test and turnaround times for diagnoses. A number of potential research applications and the increased speed of drug discovery and testing are just beginning to be explored. Microsphere technology will be improved by the availability of high-affinity antibodies and instrument calibration particles. With the current instrumentation and available antibodies, the limit of detection for cytokines, in particular, is in the low picogram and high femtogram range. The challenge is to push this limit of sensitivity for analytes that are relevant
to clinical and research applications consistently into the femtogram range. In addition, there is also a need for quality control particles that monitor fluorescence detector linearity and allow the conversion of fluorescence intensity values to molecules of equivalent soluble fluorochrome (MESF) [100]. Although these products are manufactured for conventional flow cytometers, they are not adaptable for the microspherededicated Luminex instrument. A significant advantage to multiplexing is that internal controls for the addition of the correct sample volume, detection of inhibitory immunoglobulins, nonspecific binding, and instrument performance can be monitored by including microspheres with the relevant properties [9,30,68]. Additional controls, such as those for MESF determination, could also be added to permit automated quantitation and quality control with each sample. It is anticipated that a number of companies will offer their own versions of a miniaturized flow cytometer for analysis of microsphere arrays. A high-throughput instrument that processes 600 to 1000 samples per hour has been released recently (HTS, Luminex Corp.). A fully automated instrument complete with reagent dispensing and microplate handling would maximize the potential of this technology. Undoubtedly, the microsphere chemistries will be improved to expand the capture, binding, and detection capabilities. An array that follows three or more dyes per microsphere that follows the Luminex strategy of 10 intensities of each dye (10n) would expand the size of an array into the thousands. A new spectral coding system that uses semiconductor quantum-dots (QDs), such as ZnS-capped CdSe nanocrystals, embedded into polymer beads has been described [101,102]. These QDs have narrow emission peaks and the realistic potential for 10,000 to 40,000 recognizable codes. Since most high-throughput screening assays are based on ligand coupling and/or a solid support matrix, many assay formats can be supported by fluorescence microspherebased detection. The available dyes and microsphere compositions can accommodate a variety of arrays. Microsphere-based methods should provide a rapid alternative to the methods currently used for drug susceptibility testing, phage display screening, gene expression analysis, etc., as well as the other applications discussed in the text. Once several hundred rather than 100,000 genes of interest are identified by DNA chip- or slide-based microarrays, microsphere arrays will provide a less expensive and more easily modified platform for further study of specific gene subsets. Microsphere technology holds great promise as a tool to probe both genomic and proteomic function. Acknowledgments We appreciate the assistance of J. David Taylor, Dr. Kenneth H. Pearce, and Dr. Kerry Oliver for their critical reading of the manuscript and Dr. Abbas Vafai and Dr. John G. Gray for their support of microsphere-based assay technology at the Centers for Disease Control and Prevention and GlaxoSmithKline, respectively.
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