Immunometric assay by flow cytometry using mixtures of two particle types of different affinity

Lindmo, Tore; Børmer, Ole P.; Ugelstad, J.; Nustad, Kjell

doi:10.1016/0022-1759(90)90149-p

Cited by 23 publications

(6 citation statements)

References 13 publications

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“…8,9 A number of studies have demonstrated the increased sensitivity, specificity and dynamic range obtainable with flow cytometric bead-based immunoassays over standard enzyme immunoassays. 10,11 However, a major limitation of the bead assay is the complexity and expense of flow cytometer systems.…”

Section: Introductionmentioning

confidence: 99%

Bead-based immunoassays using a micro-chip flow cytometer

et al. 2007

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A microfabricated flow cytometer has been developed for the analysis of micron-sized polymer beads onto which fluorescently labelled proteins have been immobilised. Fluorescence measurements were made on the beads as they flowed through the chip. Binding of antibodies to surface-immobilised antigens was quantitatively assayed using the device. Particles were focused through a detection zone in the centre of the flow channel using negative dielectrophoresis. Impedance measurements of the particles (at 703 kHz) were used to determine particle size and to trigger capture of the fluorescence signal. Antibody binding was measured by fluorescence at single and dual excitation wavelengths (532 nm and 633 nm). Fluorescence compensation techniques were implemented to correct for spectral overspill between optical detection channels. The data from the microfabricated flow cytometer was shown to be comparable to that of a commercial flow cytometer (BD-FACSAria).

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Section: Introductionmentioning

confidence: 99%

Bead-based immunoassays using a micro-chip flow cytometer

et al. 2007

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“…At present, latex particles are used in many applications, generally in the field of medicine and biology (7,18,19). Consequently, the ability of flow cytometry to classify latex particles with different sizes is very important.…”

Section: Classification Of Latex Particlesmentioning

confidence: 99%

Particle classification from light scattering with the scanning flow cytometer

et al. 1999

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“…The ability of the flow cytometer to discriminate between individual microspheres on the basis of size, fluorescent intensity, and/or fluorescent wavelength makes possible multianalytical assays. The use of microspheres of different sizes for multiplex assays has been described for different analytes in numerous publications (1,15,16,18,23,24). However, discrimination of microspheres by fluorescence has been documented only recently (8,14).…”

mentioning

confidence: 99%

Flow Cytometric Microsphere-Based Immunoassay: Analysis of Secreted Cytokines in Whole-Blood Samples from Asthmatics

Camilla

Mély

Magnan

et al. 2001

Clin Diagn Lab Immunol

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The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microspherebased assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-␥), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (P ‫؍‬ 0.003) and less IFN-␥ (P ‫؍‬ 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-␥ than that of atopic nonasthmatic subjects (P ‫؍‬ 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.It has been known for years that fluorescent flow cytometric detection combined with the use of sized latex microspheres allows one to perform specific and quantitative immunoassays of soluble analytes (9). The ability of the flow cytometer to discriminate between individual microspheres on the basis of size, fluorescent intensity, and/or fluorescent wavelength makes possible multianalytical assays. The use of microspheres of different sizes for multiplex assays has been described for different analytes in numerous publications (1,15,16,18,23,24). However, discrimination of microspheres by fluorescence has been documented only recently (8,14).The routine use of this attractive technology faces three distinct hurdles. First, the software commercialized with cytometers is complex and more appropriate for the qualitative cellular analysis of individual samples than for the batch mode of sampling required for the quantitative assay of several analytes. Second, reagent development faces unique analytical difficulties, such as the calibration of each individual assay in a multiplex assay and the quality of complex reagents with multiple components. Third, the concept of multiplex quantitative assays, albeit very attractive in principle, has yet to demonstrate its usefulness compared with well-acce...

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Immunometric assay by flow cytometry using mixtures of two particle types of different affinity

Cited by 23 publications

References 13 publications

Bead-based immunoassays using a micro-chip flow cytometer

Bead-based immunoassays using a micro-chip flow cytometer

Particle classification from light scattering with the scanning flow cytometer

Flow Cytometric Microsphere-Based Immunoassay: Analysis of Secreted Cytokines in Whole-Blood Samples from Asthmatics

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