Moxi GO II
™
User Guide
Page 25
for unique size discrimination (no size overlap in the size distributions) so that each unique
protein (specific to the antibody) could be detected (has its own size “channel” on the scatter
plot). As a starting point, the Bangs Labs beads
(
https://www.bangslabs.com/products/polystyrene-microspheres/functionalized-polystyrene
) in
the 4µm, 5µm, 6µm, and 7µm bead sizes should work. Beyond that, the approach would use
the classic “sandwich assay” methodology in which one would incubate the
functionalized beads with your lysed sample, wash the sample, apply a conjugated
(biotinylated) secondary antibody specific to each protein target, wash the sample, and then add
a PE-Streptavidin reporter. Note: PE is the required reporter for the system. Typically, these
steps would be performed on vacuum-filter plates or with magnetic bead systems.
As the assay is a relative quantification assay, a minimum of two tests would need to be run.
The first test would always be a “reference” or “control” sample to which all future “unknown”
samples would be compared. The Multiplex Bead Assay app is designed to walk the user
through the running of a reference sample. Once the reference data has been recorded, any
number of a samples can be compared to it.
When developing a custom multiplex assay, it is necessary to contact Orflo Technical
Support (
, 855-879-6694) to get a firmware file/setting to
correspond to the number of bead categories, target bead sizes, and expected bead
concentration.
Running a Multiplex Reference Sample
As mentioned above, the first step in running a Multiplex Assay is running a reference sample
for all other samples to be compared against. The general workflow for running a reference
sample is shown below. After selecting the “Multiplex Bead Assay” test (image below – top/left),
the user is presented with the option to “Run a Reference Bead Sample” (Image below –
top/right). Touching that icon walks the user through the standard assay (See “Running a Test
– General Instructions”). An example output for a Multiplex bead reference test is shown in the
image below – bottom/right. The blue size/categorization markers are pre-set based on a
system configuration file (in this example, beads are expected at 6µm, 8µm, 11µm and 16µm).
If the correct bead sizes are present, the system should auto-identify the clusters in region (refer
to image below - bottom/right) by:
1. Putting a red number at the top of the charge under “Beads Found.”
2. Marking the centroid of the bead cluster with a red dot.
3. List the bead concentration, MFI level for the bead in that lane/category/size-region,
and a green check mark in the black box below the scatter plot
