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Coupling Assessment
Once the coupling reaction has been completed, the coupled microspheres can be
enumerated and the efficiency of the coupling reaction assessed.
Microsphere Enumeration
Although, the protocol described in this manual will typically yield over a 90% recovery, it
is recommended that the user count the number of microspheres actually recovered after
each coupling reaction. This can be done with the use of a cell counter or hemacytometer.
Please refer to the cell counter or hemacytometer’s users manual for appropriate
instructions for doing so.
Coupling Confirmation
It is strongly recommended to assess coupling efficiency before proceeding to assay
development. The coupled microspheres can be reacted with a phycoerythrin (PE)-
labeled target or antibody that binds to the coupled protein. Alternatively, the target or
antibody may be biotinylated, then labeled with PE. This complex can then be analyzed
on a Luminex xMAP instrument. The intensity of the fluorescent signal of this reaction is
directly proportional to the amount of protein on the surface of the microspheres. This
process provides a rapid assessment of the relative amount of protein coupled to the
microspheres; however, this does not necessarily verify the functionality of the protein.
The ultimate test is the functional assay of the coupled protein.
Sample Coupling Confirmation Protocol
1. Select the appropriate antibody-coupled microsphere set or sets.
2. Resuspend the microspheres by vortex and sonication for approximately 20
seconds.
3. Prepare a working microsphere solution by diluting the coupled microsphere stocks
to a final concentration of 50 beads/μL in PBS-1% BSA.
NOTE: Up to 4 different microsphere sets may be prepared in the same mixture (in
multiplex), provided each set is a different microsphere region and the various
antibodies coupled to those regions can be detected with the same detection anti
-
body.
NOTE: At least 1mL of the microsphere solution is required for each reaction.
NOTE: Either PBS-1% BSA or PBS-BN (PBS, 1% BSA, 0.05% Azide, pH 7.4) may
be used as Assay Buffer.
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