Bio-Rad Human Metabolic and Hormone Assays - Manual (Page 4)

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Principle
Technology
The Bio-Plex ® multiplex system is built upon the three core elements of
xMAP technology:
n
Fluorescently dyed magnetic microspheres (also called beads), each
with a distinct color code or spectral address to permit discrimination of
individual tests within a multiplex suspension. This allows simultaneous
detection of up to 500 different molecules in a single well of a 96-well
microplate on the Bio-Plex
® 3D system, up to 100 different molecules
on the Bio-Plex ® 200 system, and up to 50 different molecules on the
Bio-Plex ® MAGPIX ™ system
n
A dedicated plate reader. The Bio-Plex 200 and Bio-Plex 3D systems
are flow cytometry–based instruments with two lasers and associated
optics to measure the different molecules bound to the surface of the
beads. In the Bio-Plex MAGPIX system, the sample is injected into a
chamber where the beads are imaged using LED and CCD technology
n
A high-speed digital signal processor that efficiently manages the
fluorescence data
Assay Format
The Bio-Plex Pro™ RBM metabolic and hormone assays are essentially
immunoassays formatted on magnetic beads. The assay principle is similar
to that of a sandwich ELISA (Figure 1). Capture antibodies directed against
the desired biomarker are covalently coupled to the beads. Coupled beads
react with the sample containing the biomarker of interest. After a series
of washes to remove unbound protein, a biotinylated detection antibody
is added to create a sandwich complex. The final detection complex is
formed with the addition of streptavidin-phycoerythrin (SA-PE) conjugate.
Phycoerythrin serves as a fluorescent indicator, or reporter. The use of
magnetic (MagPlex®) beads allows researchers to automate wash steps on
a Bio-Plex Pro (or similar) wash station. Magnetic separation offers greater
convenience, productivity, and reproducibility compared to vacuum filtration.
Note: The cortisol assay in Metabolic Panel 1 is a competitive
immunoassay (schematic not shown).