Bio-Rad Bio-Plex Software® Upgrades and Conversions - Manual (Page 239)

Basic Concepts
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Basic Concepts
The following is a brief overview of the basic principles of the Bio-Plex
suspension array system. For more information, see "Practical Flow
Cytometry", 3rd edition, by Howard M. Shapiro, M.D. (New York: Wiley-Liss
Inc., 1995).
Microspheres
xMAP microspheres are highly uniform, 5.5-micron polystyrene beads that
have been crosslinked during polymerization for physical and thermal stability.
Microspheres are grouped into sets; each set is then embedded with specific
quantities of two fluorescent dyes. The ratio of these two dyes gives each set
of microspheres a unique spectral address.
Each set of microspheres is then conjugated with a different reactant specific
for a particular target analyte. Reactants can include enzyme substrates,
receptors, antigens, and antibodies. A conjugated bead-reactant mixture can
be mixed with a sample to create, for example, a capture sandwich
immunoassay.
NOTE: To ensure the stability of the spectral address, it is essential to protect
the microspheres from light. Do not subject microspheres to prolonged high
temperatures, and protect them from freezing and thawing.
Reporter Fluorochromes
When you mix your samples with the microspheres, the target analytes bind to
the reactants on the surface of the beads. The amount of analyte that binds to
the beads is then quantitated through the use of a fluorochrome, R-
phycoerythrin. The intensity of this fluorochrome as detected by the array
reader is directly proportional to the amount of analyte present. The excitation
wavelength of the array reader reporter channel is 532 nm; the emission
wavelength is 575 + 12 nm. Specific information on R-phycoerythrin is shown
in the following table.
R-Phycoerythrin Specifications
Formula weight (Daltons) 240,000
Absorbance max (nm) 480, 546, 565
Extinction max (M-1cm-1) 1,960,000
Emission max (nm) 578
Quantum yield 0.82
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