A fluorescence immunoassay for soluble antigens employing flow cytometric detection

Lisi, Peter J.; Huang, Chien-Hwai; Huffman, R.A.; Teipel, John W.

doi:10.1016/0009-8981(82)90153-x

Cited by 36 publications

(17 citation statements)

References 5 publications

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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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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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“…Numerous reports on detection of snake venoms have been published and these techniques have been extensively reviewed [5][6][7][8][9]. Using particles as an assay platform is one way to replace conventional ELISA and bead-based flow cytometric immunoassays were first reported in 1982 to quantify the human antibody [10]. By using beads preconjugated to capture antibodies, the method completely removed the time for antibody immobilisation.…”

Section: Introductionmentioning

confidence: 99%

Immuno-fluorescence detection of snake venom by using single bead as the assay platform

Gao¹,

Zhang²,

Gopalakrishnakone³

2008

Journal of Experimental Nanoscience

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By using micro-scale polystyrene beads as a platform, a rapid and sensitive method for the detection of snake venom was established. In the method, antivenom antibody or BSA was covalently fixed onto the microsbead to form the capture-bead or control bead. In first step of the experiment, the venom binds to the capture-bead to form the complex through the antibody-antigen interaction. The Qdot conjugated second antibody was then added. The second antibody targeted the Qdot to the capture-bead/antigen complex and form Qdot-second antibody-antigen-capture-bead complex. This complex can be directly observed under UV-microscope. The system was applied to the testing of Naja kaouthia venom and the detection limit of this method was 5-10 ng/ml.

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“…Les premières applications de cette technologie ont concerné l'étude des cytokines [4,13], le dosage des immunoglobulines (Ig) [9], l'identification du système HLA, la sérologie bactérienne et le génotypage moléculaire [10]. Depuis peu, des réactifs sont disponibles dans le domaine de l'auto-immunité pour la détection semi-quantitative d'autoanticorps (AAC) et l'identification de leurs cibles antigéniques.…”

Section: Introductionunclassified

Démarche diagnostique en auto-immunité: apport de la technologie Multiplex. Exemple de la gamme QUANTA Plex™ (Inova)

Ghillani

Dufat

Charuel

et al. 2007

Immuno-analyse & Biologie Spécialisée

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A fluorescence immunoassay for soluble antigens employing flow cytometric detection

Cited by 36 publications

References 5 publications

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

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

Immuno-fluorescence detection of snake venom by using single bead as the assay platform

Démarche diagnostique en auto-immunité: apport de la technologie Multiplex. Exemple de la gamme QUANTA Plex™ (Inova)

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