Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP™ system

Dunbar, Sherry; Zee, Coe A. Vander; Oliver, Kerry G.; Karem, Kevin L.; Jacobson, James W.

doi:10.1016/s0167-7012(03)00028-9

Cited by 264 publications

(184 citation statements)

References 9 publications

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“…However, evaluation of user-determined protein targets can be achieved by creating appropriate specific probes sets (de Jager et al, 2003) using commercially available bead coupling kits (e.g., Amine Coupling Kit (#171-406001) from BioRad). Furthermore, bead-based assays can be designed, not only using immunoassays, but also around enzyme, receptor ligand and DNA hybridization capture molecules, further extending their utility (Dunbar et al, 2003;Earley et al, 2002). Gallagher and Appenzeller (1999) suggest that the predominant approach to scientific inquiry has been reductionist.…”

Section: Discussionmentioning

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%

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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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Section: Discussionmentioning

confidence: 99%

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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“…A similar pattern of Bfra3 and Bdis3 response (data not shown) was seen for high numbers of B. fragilis loaded onto membrane filters (≥6 ×10 5 cells), perhaps from low levels of contamination or intraspecies variability, particularly with a relatively low hybridization temperature of 53° C. However, no response to Bdis3 was observed for lower numbers of B. fragilis (≤6×10 4 cells). Cross-hybridization with high concentrations of target also has been observed in other Luminex assays (Dunbar et al, 2003).…”

Section: Luminex Verification Through Specificity Testing With Enviromentioning

confidence: 74%

“…For sensitivity experiments, the cMFI value was reported to facilitate visual interpretation of results. Otherwise, values of MFI were reported to show the intensity of fluorescence for comparison to other work (Dunbar et al, 2003).…”

Section: Hybridization Of Amplicons To Luminex Probesmentioning

confidence: 99%

“…The Luminex system has primarily been used for clinical applications (Dunbar et al, 2003;Dunbar, 2006), although some environmental applications have been reported (Ellison and Burton, 2005;Spiro et al, 2000;Spiro and Lowe, 2002). Any molecule or chemical group that can be recognized by reactive or complementary functional groups can be immobilized to the surface of the microspheres.…”

Section: Introductionmentioning

confidence: 99%

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Luminex detection of fecal indicators in river samples, marine recreational water, and beach sand

Baums

Goodwin

Kiesling

et al. 2007

Marine Pollution Bulletin

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Research to understand and remediate coastal pollution is moving toward a multitiered approach in which traditional enumeration of fecal indicators is accompanied by molecular analysis of a variety of targets. Technology that rapidly detects multiple microbial contaminants would benefit from such an approach. The Luminex ® 100™ system is a suspension array that assays multiple analytes rapidly in a single well of a microtiter plate. The ability of the system to simultaneously detect multiple fecal indicating bacteria in environmental samples was tested. Primer/probe sets were designed to simultaneously detect the following fecal indicators: the Bacteroides fragilis group, Enterococcus spp., Escherichia coli & Shigella spp., B. distasonis, and Ent. faecalis. Specificity and sensitivity of the Luminex probes was tested against laboratory cultures. In addition, sequencing, culture plate testing, and specificity testing with environmental isolates were steps taken to validate the function of the assay with environmental samples. Luminex response to cultures and to environmental samples was consistent with sequencing results, suggesting that the technology has the potential to simultaneously detect multiple targets for coastal water quality applications, particularly as progress is made to efficiently extract DNA from water and sediment matrices.

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Multiplexed Detection of Fungal Nucleic Acid Signatures

2008

Self Cite

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Diagnoses of opportunistic mycotic infections constitute an increasing clinical problem. Conventional diagnostic tests are time consuming and lack specificity and sensitivity for accurate and timely prognoses. This unit provides a comprehensive description of a fungal detection method that combines nucleic acid signatures with flow cytometry. The multiplexed assay, which uses xMAP technology, consists of unique fluorescent microspheres covalently bound to species‐specific fungal oligonucleotide probes. In the presence of the complementary target sequence, the probe hybridizes to its biotinylated target. Quantification of the reaction is based on the fluorescence signal of the reporter molecule that binds to the biotin moieties of the target. The assay can be expanded to include other microorganisms and has the capability to simultaneously test 100 different fungal probes per tube/well. The speed, flexibility in design, and high‐throughput capability makes this assay an attractive diagnostic tool for fungal infections and other related maladies. Curr. Protocol. Cytom. 44:13.9.1‐13.9.21. © 2008 by John Wiley & Sons, Inc.

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Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP™ system

Cited by 264 publications

References 9 publications

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

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

Luminex detection of fecal indicators in river samples, marine recreational water, and beach sand

Multiplexed Detection of Fungal Nucleic Acid Signatures

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