Luminex 100 IS Developer Workbench Guide Version 2.3 - Manual (Page 10)

Luminex 100 IS Developer Workbench Guide Version 2.3
x
MAP Technology
4 PN 89-00002-00-084 Rev. B
and do not exceed a bead concentration of 2.5 x 10
6
microspheres/
mL.
Repetitive xMAP
Microsphere
Measurements
The reporter, bound to reagents at the surface of the xMAP
microspheres, provides raw analytical data. Each xMAP microsphere
of a particular signal theoretically binds an equal number of reporter
molecules. Thus, an xMAP microsphere suspension provides near-
liquid-phase reaction kinetics. Equal binding results in a statistically
even distribution of reporter on each xMAP microsphere in a set.
Therefore, when the Luminex 100 IS system 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 xMAP 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 contains dyes that 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 detects fluorescence bound to the surface of each xMAP
microsphere, and each reporter emission quantitates each of the
distinct assays.
Table 2 displays acceptable reporter fluorochromes wavelengths
excitation/emission.
Sample Volume
Sample volumes or sample sizes range from 20 to 200 µL. Ensure
that some sample remains in the well after aspiration; about 25 µL
greater than the sample volume. This amount may vary depending on
the type of plate used. After acquisition, the Luminex 100 analyzer
washes the sample lines resulting in ejection of approximately 160
µL of sheath fluid back into the well. Ensure that there is room to
add this amount to the well without overflowing 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