Luminex 100 IS Version 2.1 - Manual (Page 12)

Luminex 100 IS Developer Guide to xMAP Technology Version 2.1 x MAP Technology
6 PN 89-00002-00-062 Rev. A
Repetitive xMAP
Microsphere
Measurements
The reporter, bound to reagents at the surfaces of the xMAP
microspheres, provides raw analytical data. Because an xMAP
microsphere suspension provides near liquid phase reaction kinetics,
each microsphere of a particular signal theoretically binds an equal
number of reporter molecules. Equal binding results in a statistically
even distribution of reporter on each microsphere in a set. Therefore,
when the Luminex 100 IS registers numerous events for each set, the
system reports individual population values based on an even coating
of reporter. This means that numerous replicates for each
microsphere population are measured from a single reaction vessel.
The confidence in a given measurement strengthens with increased
replicate measurement. The median value for the microspheres
having a specific signal is reported as the result.
Classification and
Reporter Fluorochromes
Each xMAP microsphere is dyed to emit light in a certain
classification channel. All xMAP microspheres of a given emission
represent a distinct assay within a multiplex of assays. A reporter
channel is used to detect fluorescence bound to the surface of each
xMAP microsphere, and each reporter emission quantitates each of
the distinct assays.
The following chart displays acceptable reporter fluorochromes
wavelengths excitation/emission.
Sample Size
Sampling volume or sample size can range from 20 to 200
microliters. Assays must have enough volume in the wells so that
after this sampling volume is taken up, some sample remains. Make
the volume in the wells at least 25 uL greater than the sample
volume. This amount may vary depending on the type of plate used.
After acquisition, the instrument washes the sample lines resulting in
ejection of approximately 160 microliters of sheath fluid back into
the well. Make sure that there is room to add this amount to the well
without overflowing the well and contaminating other wells.
Table 2: Reporter Fluorochromes
R-Phycoerythrin Alexa 532
Formula weight (Daltons) 240,000 470
Absorbance max (nm) 480, 546, 565 531
Extinction max (M
-1
cm
-1
)
1,960,000 83,800
Emission max (nm) 578 554
Quantum yield 0.82 0.8