Fluorescence single-molecule counting assays for high-sensitivity detection of cytokines and chemokines

By reducing interrogation zones to these dimensions and carefully tuning the timescales of the data acquisition and molecular flow rates, background interference is decreased and individual molecules are readily recorded as discrete fluorescence bursts above background (6). Immune complexes were for...

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Bibliographic Details
Published in:Clinical chemistry (Baltimore, Md.) Md.), 2007-11, Vol.53 (11), p.2010-2012
Main Authors: Qui, Haoqun, Ferrell, Evan P, Nolan, Niamh, Phelps, Bruce H, Tabibiazar, Raymond, Whitney, Duncan H, Naelfski, Eric A
Format: Article
Language:English
Subjects:
Calibration
Chemokine CCL2 - blood
Comparative analysis
Cytokines
Cytokines - blood
Data acquisition
Disease management
Flow rates
Fluorescence
Humans
Immunoassay - methods
Interferon-gamma - blood
Molecular weight
Monoclonal antibodies
Proteins
Tumor Necrosis Factor-alpha - blood
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Summary:By reducing interrogation zones to these dimensions and carefully tuning the timescales of the data acquisition and molecular flow rates, background interference is decreased and individual molecules are readily recorded as discrete fluorescence bursts above background (6). Immune complexes were formed in MultiScreen HTS, BV microfiltration plates (Millipore) in 3 successive steps (capture of analyte to microparticles, binding of captured antigen with biotinylated detection antibodies, and binding of SA-A647 to bound detection antibodies) separated by removal of unbound material with microfiltration on a MultiScreen HTS vacuum manifold (Millipore) and extensive washing. Calibration curves from SMIAs for tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and monocyte chemotactic protein (MCP)-1 performed on 3 separate occasions under identical conditions are provided in Fig.
ISSN:0009-9147
1530-8561
DOI:10.1373/clinchem.2007.091306