Bio-Rad Bio-Plex Pro Human Angiogenesis Reagent and Diluent Kits - 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 angiogenesis target is covalently coupled to internally
dyed beads. The coupled beads are allowed to react with a sample
containing the angiogenesis target. 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 angiogenesis target. 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.
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