Multiplexed Microsphere‐Based Flow Cytometric Assays

Kellar, Kathryn L.; Iannone, Marie A.

doi:10.1002/chin.200338280

Cited by 62 publications

(82 citation statements)

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“…Results were consistently reproduced for numerous samples and synthetic probes independently conjugated to microspheres. Surface carboxylated microsphere sets with distinct emission spectra and intensities which enable the concurrent analyses of hundreds or more target sequences are available from several manufacturers [Kellar and Iannone, 2002]. Carbodiimide coupling is a robust chemistry for probe attachment that has been standardized with routine laboratory protocols [Dunbar et al, 2003;Fulton et al, 1997].…”

Section: Discussionmentioning

confidence: 99%

Determination of genomic copy number with quantitative microsphere hybridization

et al. 2006

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Communicated by Jing ChengWe developed a novel quantitative microsphere suspension hybridization (QMH) assay for determination of genomic copy number by flow cytometry. Single copy (sc) products ranging in length from 62 to 2,304 nucleotides [Rogan et al., 2001;Knoll and Rogan, 2004] from ABL1 (chromosome 9q34), TEKT3 (17p12), PMP22 (17p12), and HOXB1 (17q21) were conjugated to spectrally distinct polystyrene microspheres. These conjugated probes were used in multiplex hybridization to detect homologous target sequences in biotinylated genomic DNA extracted from fixed cell pellets obtained for cytogenetic studies. Hybridized targets were bound to phycoerythrin-labeled streptavidin; then the spectral emissions of both target and conjugated microsphere were codetected by flow cytometry. Prior amplification of locus-specific target DNA was not required because sc probes provide adequate specificity and sensitivity for accurate copy number determination. Copy number differences were distinguishable by comparing the mean fluorescence intensities (MFI) of test probes with a biallelic reference probe in genomic DNA of patient samples and abnormal cell lines. Concerted 5 0 ABL1 deletions in patient samples with a chromosome 9;22 translocation and chronic myelogenous leukemia were confirmed by comparison of the mean fluorescence intensities of ABL1 test probes with a HOXB1 reference probe. The relative intensities of the ABL1 probes were reduced to 0.5970.02 fold in three different deletion patients and increased 1.4270.01 fold in three trisomic 9 cell lines. TEKT3 and PMP22 probes detected proportionate copy number increases in five patients with Charcot-Marie-Tooth Type 1a disease and chromosome 17p12 duplications. Thus, the assay is capable of distinguishing one allele and three alleles from a biallelic reference sequence, regardless of chromosomal context. Hum Mutat 27(4), [376][377][378][379][380][381][382][383][384][385][386] 2006.

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Section: Discussionmentioning

confidence: 99%

Determination of genomic copy number with quantitative microsphere hybridization

et al. 2006

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“…Samples and positive controls exhibiting a coefficient of variation (CV) >10% were omitted from final data analyses (Kellar and Iannone, 2002). Furthermore, all signals were normalized by inclusion of fluorescence from duplicate blank wells that consisted initially of only Cell Lysis Buffer (50 μl per well) for tissue and sample diluent (50 μl per well) for serum.…”

Section: Cytokine Measurementsmentioning

confidence: 99%

“…Recent availability of commercial instruments based on bead-based immunoassay technology (Dasso et al, 2002;de Jager et al, 2003;Dunbar et al, 2003;Earley et al, 2002;Kellar and Iannone, 2002;Kellar et al, 2001;Prabhakar et al, 2002;Vignali, 2000) is likely to help remove this void. For example, the Bio-Plex™ Suspension Array system (Bio-Rad; Hercules, CA) is an easy-to-use and flexible unit capable of simultaneously analyzing up to 100 (6-150 kDa) proteins from as little as 12 μl of sample, which has been done for analyses of serum (de Jager et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue

Hulse

Kunkler

Fedynyshyn

et al. 2004

Journal of Neuroscience Methods

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The ability to simultaneously quantify multiple signaling molecule protein levels from microscopic neural tissue samples would be of great benefit to deciphering how they affect brain function. This follows from evidence that indicates signaling molecules can be pleiotropic and can have complex interactive behavior that is regionally and cellularly heterogeneous. Multiplexed examination of tissue proteins has been exceedingly difficult because of the absence of available techniques. This void now has been removed by the commercial availability of bead-based immunoassays for targeted proteins that allow analyses of up to 100 (6-150 kDa) proteins from as little as 12 μl. Thus far used only for sera (human and mouse) and culture media, we demonstrate here that sensitive (as low as 2 pg/ml), wide-ranging (up to 2-32 000 pg/ml), accurate (8% intra-assay covariance) and reliable (4-7% inter-assay covariance) measurements can be made of nine exemplary cytokines (e.g., IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF-α) simultaneously not only from rat serum but, for the first time, also brain tissue. Furthermore, we describe animal handling procedures that minimize stress as determined by serum glucocorticoid levels since they can influence cytokine expression.

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“…The fields of application include techniques used in microbiology, like immunoassays of bacterial (9) or viral (10) antigens, or analysis of antiviral/ antibacterial antibodies (7,11), both groups of molecules can be a diagnostic aid for such infections. While this approach matches conventional methods in sensitivity, reproducibility, and simplicity, it seems to have significant advantages by virtue of the possibility for multiplex analysis (5,6,9,(11)(12)(13)(14)(15). Furthermore, sequence-specific capture of PCR-amplified genomic or cDNA sequences allows the detection of single nucleotide polymorphisms (16)(17)(18), and also turns this platform into a possible alternative to microarrays on chips to be used for the characterization of gene expression profiles (19).…”

Section: Introductionmentioning

confidence: 99%

Biological microbeads for flow‐cytometric immunoassays, enzyme titrations, and quantitative PCR

Pataki

Szabó²,

Lantos

et al. 2005

Cytometry Pt A

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BackgroundIntroduction of microbeads into flow‐cytometry has created a new scenario, making quantitative measurement of molecules dispersed in a homogeneous phase, with an extremely wide realm of already realized and potential applications possible. Development of this field has lead to specialized instrumentation and microbead arrays, dedicated to certain applications.MethodsFormaldehyde‐fixed yeast and bacterial cells were conjugated with avidin and applied as microbeads, to establish a simple, convenient, flexible, and inexpensive flow‐cytometric platform for various immunological and biochemical assays.ResultsWe have tested these “biological microbeads” for the simultaneous titration of human α‐fetoprotein (AFP) and human Chorionic Gonadotropin (βhCG) hormone levels, for the titration of proteolytic and nucleolytic (restriction) enzymes, and for quantitative PCR, using biotinylated and fluorescent primers.ConclusionsThe use of biological microbeads for various immunological and biochemical assays has been demonstrated. The flow‐cytometric methods proved to be at least as sensitive as the standard biochemical or immunological tests. For proteinase K activity measurements, a single enzyme molecule in the sample could be detected. The sensitivity, versatility, and low cost of the assays may advance flow‐cytometry to become a central methodological platform in most laboratories. The biological microbeads offer virtually unlimited possibilities for fluorescent labeling (addressing), conjugation of ligand binding molecules, and they are easy to handle and perform well in a multiplex format. © 2005 International Society for Analytical Cytology

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Multiplexed Microsphere‐Based Flow Cytometric Assays

Abstract: For Abstract see ChemInform Abstract in Full Text.

Cited by 62 publications

References 1 publication

Determination of genomic copy number with quantitative microsphere hybridization

Determination of genomic copy number with quantitative microsphere hybridization

Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue

Biological microbeads for flow‐cytometric immunoassays, enzyme titrations, and quantitative PCR

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