Bio-Rad Bio-Plex® Precision Pro™ Human Cytokine Assays - Manual (Page 4)

Section 2
Principle
Technology
The Bio-Plex
® suspension array system is built around three core
technologies. The first is a novel technology that uses up to 100 unique
fluorescently dyed beads (xMAP technology) that permit the simultaneous
detection of up to 100 different types of molecules in a single well of a
96-well microplate. The second is a flow cytometer with two lasers and
associated optics to measure the different molecules bound to the
surface of the beads. The third is a high-speed digital signal processor
that efficiently manages the fluorescent output.
Assay Format
The principle of these 96-well plate-formatted, bead-based assays is similar
to a capture sandwich immunoassay. An antibody directed against the
desired target cytokine is covalently coupled to internally dyed beads. The
coupled beads are allowed to react with a sample containing the target
cytokine. After a series of washes to remove unbound protein, a
biotinylated detection antibody specific for a different epitope is added to
the reaction. The result is the formation of a sandwich of antibodies around
the target cytokine. Streptavidin-phycoerythrin (streptavidin-PE) is then
added to bind to the biotinylated detection antibodies on the bead surface.
Data Acquisition and Analysis
Data from the reaction are then acquired using the Bio-Plex suspension
array system (or Luminex system), a dual-laser, flow-based microplate
reader system. The contents of the well are drawn up into the reader.
The lasers and associated optics detect the internal fluorescence of the
individual dyed beads as well as the fluorescent signal on the bead
surface. This identifies each assay and reports the level of target protein
in the well. Intensity of fluorescence detected on the beads indicates the
relative quantity of targeted molecules. A high speed-digital processor
efficiently manages the data output, which is further analyzed and
presented as fluorescence intensity on Bio-Plex Manager™ software, the
accompanying software package.
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