Bio-Rad Bio-Plex Pro Human Acute Phase Reagent and Diluent Kits - Manual (Page 6)

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 protein is covalently coupled to internally dyed beads. The
coupled beads are allowed to react with a sample containing the target
protein. 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
protein. Streptavidin-phycoerythrin (streptavidin-PE) is then added to bind to
the biotinylated detection antibodies on the bead surface for flourescence-
based detection.
Data Acquisition and Analysis
Data from the reaction are then acquired using the Bio-Plex suspension
array system, 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 that
corresponds to the amount of detected target protein. 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.
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