Multiplex microsphere‐based flow cytometric platforms for protein analysis and their application in clinical proteomics – from assays to results

Hsu, Hsin-Yun; Joos, Thomas; Koga, Hisashi

doi:10.1002/elps.200900211

Cited by 60 publications

(46 citation statements)

References 88 publications

(78 reference statements)

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“…The intended use should be the driving force on selecting the appropriate multiplex assay. In addition, several recent publications comprehensively review the latest multiplexing technologies (5)(6)(7)(8).…”

Section: Overview Of Multiplexing Technologiesmentioning

confidence: 99%

Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development

Jani

Allinson²,

Berisha

et al. 2015

AAPS J

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Abstract. Multiplex ligand binding assays (LBAs) are increasingly being used to support many stages of drug development. The complexity of multiplex assays creates many unique challenges in comparison to single-plexed assays leading to various adjustments for validation and potentially during sample analysis to accommodate all of the analytes being measured. This often requires a compromise in decision making with respect to choosing final assay conditions and acceptance criteria of some key assay parameters, depending on the intended use of the assay. The critical parameters that are impacted due to the added challenges associated with multiplexing include the minimum required dilution (MRD), quality control samples that span the range of all analytes being measured, quantitative ranges which can be compromised for certain targets, achieving parallelism for all analytes of interest, cross-talk across assays, freeze-thaw stability across analytes, among many others. Thus, these challenges also increase the complexity of validating the performance of the assay for its intended use. This paper describes the challenges encountered with multiplex LBAs, discusses the underlying causes, and provides solutions to help overcome these challenges. Finally, we provide recommendations on how to perform a fit-forpurpose-based validation, emphasizing issues that are unique to multiplex kit assays.

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Section: Overview Of Multiplexing Technologiesmentioning

confidence: 99%

Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development

Jani

Allinson²,

Berisha

et al. 2015

AAPS J

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“…As a result, they are widely used for performing chemical or biological assays in the biochemistry, biophysics, medicine, and life science fields Tsai et al 2007;Chen and Wang 2008;Lin et al 2009;Fercher et al 2009;Hong et al 2010;Tran et al 2010;Li et al 2011). Among the various microfluidic devices available, flow cytometers provide a high-throughput means of sorting and counting single cells, and are used extensively in the hematology, immunology, and microbiology fields (Lin and Lee 2008;Fu et al 2008;Tsai et al 2008;Chen and Wang 2009;Zhang et al 2009;Hou et al 2009;Hsu et al 2009;Wang et al, 2010;Xuan et al, 2010). In traditional flow cytometer, it can be used for analyzing signals at more than one fluorescence wavelength and light scatter, a rough measurement of size and density.…”

Section: Introductionmentioning

confidence: 99%

Microflow cytometer incorporating sequential micro-weir structure for three-dimensional focusing

Lee

Hou

Yang

et al. 2011

Microfluid Nanofluid

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A novel microflow cytometer is proposed in which the sample stream is focused initially in the horizontal (X-Y) plane by two sheath flows and is then focused in the vertical (Y-Z) plane by a sequential micro-weir structure before entering the detection region of the device. The proposed device is fabricated using a modified glass fabrication process, and is then characterized both numerically and experimentally using fluorescent polystyrene beads with diameters of 7 and 15 lm, respectively. The experimental and numerical results confirm the effectiveness of the hydrodynamic sheath flows in centralizing the particles in the horizontal plane prior to entering the sequential micro-weir structure. Furthermore, it is shown numerically that the micro-weir structure confines the particle stream to the center of the vertical plane such that the particles pass through the detection region in a oneby-one fashion can be counted with a high degree of reliability. The experimental results confirm that the proposed 3-D focusing scheme enables the fluorescent beads with different diameters to be reliably sorted and counted.

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“…Assays for interrogating binding interactions which use trapped microbeads with bound probes in a microfluidic cell are similar to flow cytometric assays in which beads with surface probes are suspended in a medium with a fluorescently labeled target analyte and binding interactions are identified by passing the microbeads through a flow cytometer to identify beads fluorescing with targets. 4,[14][15][16] The microbead microfluidic format has the advantage of reduced consumptions of analytes and reagents, higher probe densities and the versatility to identify the probes on the probe surface without encoding. Flow cytometry requires encoding the beads with a fluorescent label to identify the probe on the microbead surface.…”

Section: Introductionmentioning

confidence: 99%

Transport of biomolecules to binding partners displayed on the surface of microbeads arrayed in traps in a microfluidic cell

2017

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Arrays of probe molecules integrated into a microfluidic cell are utilized as analytical tools to screen the binding interactions of the displayed probes against a target molecule. These assay platforms are useful in enzyme or antibody discovery, clinical diagnostics, and biosensing, as their ultraminiaturized design allows for high sensitivity and reduced consumption of reagents and target. We study here a platform in which the probes are first grafted to microbeads which are then arrayed in the microfluidic cell by capture in a trapping course. We examine a course which consists of V-shaped, half-open enclosures, and study theoretically and experimentally target mass transfer to the surface probes. Target binding is a two step process of diffusion across streamlines which convect the target over the microbead surface, and kinetic conjugation to the surface probes. Finite element simulations are obtained to calculate the target surface concentration as a function of time. For slow convection, large diffusive gradients build around the microbead and the trap, decreasing the overall binding rate. For rapid convection, thin diffusion boundary layers develop along the microbead surface and within the trap, increasing the binding rate to the idealized limit of untrapped microbeads in a channel. Experiments are undertaken using the binding of a target, fluorescently labeled NeutrAvidin, to its binding partner biotin, on the microbead surface. With the simulations as a guide, we identify convective flow rates which minimize diffusion barriers so that the transport rate is only kinetically determined and measure the rate constant. Published by AIP Publishing.

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Multiplex microsphere‐based flow cytometric platforms for protein analysis and their application in clinical proteomics – from assays to results

Cited by 60 publications

References 88 publications

Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development

Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development

Microflow cytometer incorporating sequential micro-weir structure for three-dimensional focusing

Transport of biomolecules to binding partners displayed on the surface of microbeads arrayed in traps in a microfluidic cell

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