4 Notes
1. All the washing steps described in the procedures are carried
out on a 96-plate magnet. For that, add the washing buffer off
the magnet. Then, put the plate on the magnet, adjust the clip,
and let the beads soak for 1 min. Then, invert the plate attached
to the magnet to remove the liquid. Repeat the process as many
times as it is specified. Magnets for 384-well plates are also
available (such as VP 771IAAZM-1, V&P Scientific Inc).
Washing steps can also be implemented on automated plate
washers suited for handling magnetic beads (such as EL406,
Biotek).
2. EDC and NHS should be equilibrated to room temperature
before opening the vials. Since both are highly hygroscopic
substances, failure to allow equilibrating the vials to room
temperature might reduce the coupling efficiency.
3. Other fluorescent dyes than R-phycoerythrin such as Alexa546,
Alexa532, or Cy3 can be utilized as well but Luminex Corp. has
indicated that lower reporter signal intensities can be observed.
Different suppliers for R-phycoerythrin can also be compared
to achieve a desired assay performance.
4. HBR6 contains purified murine immunoglobulins with specific
binders that neutralize by active attachment to the heterophilic
antibody. Upon received, store the HBR6 at or below À20
C.
Thaw the HBR at 4
C before use and mix the reagent well by
gentle inversion ten times. Do not foam the reagent. Add the
HBR6 directly to the assay buffer so that each tube of supplemented assay buffer can be used at a rate of 40 mg of HBR6 per
sample. In order to avoid freeze–thaw cycles, prepare aliquots
for next usages of HBR6 and store them at À20
C.
5. Avoid protein stabilizing agents, Tris, or other amine-based
antibody solution buffers as they reduce the coupling efficiency.
6. At all times, try to minimize the light exposure, especially to
direct sunlight, as the internal fluorescence of the beads as well
as reporter fluorophores could be bleached. During incubation, protect the plates with an opaque cover or place plate into
a light-tight box.
7. Do not interrupt the process after dissolving EDC and NHS, as
these substances are susceptible to hydrolysis upon being dissolved. Delays in the procedure until EDC and NHS have been
added might lead to a reduced coupling efficiency.
8. Adapt the instrumentation setting according to the bead IDs
included in the bead array and count at least 50 beads per bead
ID. We suggest the “median fluorescence intensity” (MFI) as
output for further data processing and analysis. To evaluate if
Minimizing Rheumatoid Factor Interference
149
1. All the washing steps described in the procedures are carried
out on a 96-plate magnet. For that, add the washing buffer off
the magnet. Then, put the plate on the magnet, adjust the clip,
and let the beads soak for 1 min. Then, invert the plate attached
to the magnet to remove the liquid. Repeat the process as many
times as it is specified. Magnets for 384-well plates are also
available (such as VP 771IAAZM-1, V&P Scientific Inc).
Washing steps can also be implemented on automated plate
washers suited for handling magnetic beads (such as EL406,
Biotek).
2. EDC and NHS should be equilibrated to room temperature
before opening the vials. Since both are highly hygroscopic
substances, failure to allow equilibrating the vials to room
temperature might reduce the coupling efficiency.
3. Other fluorescent dyes than R-phycoerythrin such as Alexa546,
Alexa532, or Cy3 can be utilized as well but Luminex Corp. has
indicated that lower reporter signal intensities can be observed.
Different suppliers for R-phycoerythrin can also be compared
to achieve a desired assay performance.
4. HBR6 contains purified murine immunoglobulins with specific
binders that neutralize by active attachment to the heterophilic
antibody. Upon received, store the HBR6 at or below À20
C.
Thaw the HBR at 4
C before use and mix the reagent well by
gentle inversion ten times. Do not foam the reagent. Add the
HBR6 directly to the assay buffer so that each tube of supplemented assay buffer can be used at a rate of 40 mg of HBR6 per
sample. In order to avoid freeze–thaw cycles, prepare aliquots
for next usages of HBR6 and store them at À20
C.
5. Avoid protein stabilizing agents, Tris, or other amine-based
antibody solution buffers as they reduce the coupling efficiency.
6. At all times, try to minimize the light exposure, especially to
direct sunlight, as the internal fluorescence of the beads as well
as reporter fluorophores could be bleached. During incubation, protect the plates with an opaque cover or place plate into
a light-tight box.
7. Do not interrupt the process after dissolving EDC and NHS, as
these substances are susceptible to hydrolysis upon being dissolved. Delays in the procedure until EDC and NHS have been
added might lead to a reduced coupling efficiency.
8. Adapt the instrumentation setting according to the bead IDs
included in the bead array and count at least 50 beads per bead
ID. We suggest the “median fluorescence intensity” (MFI) as
output for further data processing and analysis. To evaluate if
Minimizing Rheumatoid Factor Interference
149