A Generic Particle-Based Nonradioactive Homogeneous Multiplex Method for High-Throughput Screening Using Microvolume Fluorimetry

Martens, Christine L.; Bakker, Alice; Rodríguez, Ana B.; Mortensen, Richard B.; Barrett, Ronald W.

doi:10.1006/abio.1999.4184

Cited by 29 publications

(14 citation statements)

References 16 publications

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“…The relative standard deviation data in this work were similar to those reported for some other microsphere-and microspot-based fluorescent sensing methods (14,21,22,48,49). For instance, 15-25% microsphere-to-microsphere CV values were found in fluorometric microvolume assays (48,49).…”

Section: Resultssupporting

confidence: 87%

“…For instance, 15-25% microsphere-to-microsphere CV values were found in fluorometric microvolume assays (48,49). In future work, the precision of our microsphere-based assays could be increased by using arrays with higher microsphere density, improved immobilization chemistry, and optical imaging fibers and an imaging system better suited for these analyses.…”

Section: Resultsmentioning

confidence: 97%

“…The results of our experiments hold promise for the development of further randomly ordered immunoarrays. Recently, several microparticle-based multiplexed biomedical immunoanalyses and receptor binding assays have been devised (7,12,14,21,48,50). The technique should also be useful in other fields of applications including environmental monitoring as well as industrial process and quality control.…”

Section: Resultsmentioning

confidence: 98%

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A Duplexed Microsphere-Based Fluorescent Immunoassay

Szurdoki

Michael

Walt

2001

Analytical Biochemistry

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 98%

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A Duplexed Microsphere-Based Fluorescent Immunoassay

Szurdoki

Michael

Walt

2001

Analytical Biochemistry

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“…The microvolume fluorimetry assay, measuring ligand-dependent association of fluorescently labeled IL-5R␣ ECD with unlabeled bead-bound receptor, was performed using an fluorometric microvolume assay technology (FMAT) instrument (Perkin-Elmer) as described previously (31). Gel filtration chromatography was performed in 50 mM Tris (pH 7.5)͞0.5 M NaCl on a Pharmacia Smart system using a Precision Superdex 200 3.2͞30 column at 25°C with a flow rate of 10 l͞min.…”

Section: Methodsmentioning

confidence: 99%

“…Several different methods were used to investigate whether AF18748 was similarly able to bind to two IL-5R␣ chains. First, a ''sandwich-'' binding assay was devised using microvolume fluorimetry (31) to measure the liganddependent association of two receptor ␣ chains. IL-5R␣ ECD was immobilized onto 10-m beads and incubated with soluble, fluorescently labeled IL-5R␣ ECD in the presence or absence of IL-5 or peptide.…”

Section: Identification Of Il-5 Antagonist Peptides By Peptide Librarmentioning

confidence: 99%

A potent dimeric peptide antagonist of interleukin-5 that binds two interleukin-5 receptor α chains

England

Palaniappan

Uings

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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Two series of peptides that specifically bind to the extracellular domain of the ␣ chain of the human interleukin-5 receptor (IL-5R␣), but share no primary sequence homology to IL-5, were identified from libraries of random recombinant peptides. Affinity maturation procedures generated a 19-aa peptide that binds to the IL-5 receptor ␣͞␤ heterodimer complex with an affinity equal to that of IL-5 and is a potent and specific antagonist of IL-5 activity in a human eosinophil adhesion assay. The active form of the peptide is a disulfide-crosslinked dimer that forms spontaneously in solution. Gel filtration analysis, receptor-binding studies, and analytical ultracentrifugation reveal that the dimeric peptide binds simultaneously to two receptor ␣ chains in solution. Furthermore, the dimer peptide, but not IL-5, can activate a chimeric receptor consisting of the IL-5R␣ extracellular domain fused to the intracellular domain of the epidermal growth factor receptor, thus demonstrating that the peptide also promotes receptor dimerization in a cellular context. The functional antagonism produced by the bivalent interaction of the dimeric peptide with two IL-5R ␣ chains represents a distinctive mechanism for the antagonism of cytokines that use heteromeric receptors.I nterleukin-5 (IL-5) is a T cell-derived hematopoietic cytokine that acts exclusively on cells of the eosinophil and basophil lineage (1, 2). Although IL-5 can regulate many of the functions of mature, terminally differentiated eosinophils such as cell survival (3), adhesion (4), and activation (5), its primary function is to promote the differentiation and expansion of eosinophil precursors in the bone marrow (6).Transgenic mice that constitutively overexpress IL-5 in their lungs exhibit systemic and airway eosinophilia, bronchial hyperreactivity, and histopathological features characteristic of human asthma (7). Administration of recombinant IL-5 into the airways of either guinea pigs (8) or mildly asthmatic individuals (9) promotes a lung eosinophilia and bronchial hyperreactivity. Moreover, blocking the activity of IL-5 through administration of neutralizing anti-IL-5 antibodies (10-12), or through IL-5 gene deletion (13), prevents allergen-induced airway eosinophilia and bronchial hyperreactivity. Together, these data demonstrate that IL-5 plays a central role in the development of allergen-induced eosinophilia in animals and suggest that an anti-IL-5 therapeutic could provide an alternative approach to the treatment of human asthma perhaps more specific than current anti-inf lammatory therapies such as inhaled corticosteroids.The IL-5 receptor is a heterodimer consisting of an ␣ chain that specifically binds IL-5 and a signal-transducing ␤ c chain shared with the receptors for two structurally related cytokines: granulocyte-macrophage colony-stimulating factor (GM-CSF) and . A number of protein-based IL-5 antagonists have been reported, including soluble IL-5 receptor ␣ chains (IL5R␣s) that sequester the ligand in solution (15), single point mutants of IL...

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Ultra Sensitive Bioaffinity Assay for Micro Volumes

et al. 2000

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An ultrasensitive separation free micro volume bioaffinity assay method is presented. The concept is based on the use of two-photon excited fluorescence and polystyrene microspheres as bioaffinity binding solid phase. Measurements are performed by observing individual microspheres in the diffraction limited focal volume of twophoton excitation.The method is suitable for measuring bioaffinity binding assays directly from the reaction volume without separation and washing steps. Depending on the sensitivity requirements the assay can be scaled. Scaling is achieved by adjusting the amount of solid phase microspheres for the assay. The scalability is demonstrated by measuring standard curves for an immunoassay. As a model assay we chose the human alpha-fetoprotein (AFP) immunometric assay. Operating ranges from 10 pg/ml up to 20 ng/ml and 100 pg/ml up to 400 ng/ml were achieved.

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A Generic Particle-Based Nonradioactive Homogeneous Multiplex Method for High-Throughput Screening Using Microvolume Fluorimetry

Cited by 29 publications

References 16 publications

A Duplexed Microsphere-Based Fluorescent Immunoassay

A Duplexed Microsphere-Based Fluorescent Immunoassay

A potent dimeric peptide antagonist of interleukin-5 that binds two interleukin-5 receptor α chains

Ultra Sensitive Bioaffinity Assay for Micro Volumes

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