of the samples. To ensure successful antibody coupling, evaluate the
immobilization efficiency through the use of fluorescently labeled
anti-species-specific antibodies.
1. Prepare the bead stock to a final concentration of 100 beads/
ID/μL.
2. Vortex the bead stock solution and sonicate for 5 min.
3. Distribute 5μL of bead mixture to each well of an assay plate.
4. Prepare the detection secondary antibody solutions described
above and add 50μL of these solutions to the wells with bead
mixture.
5. Incubate for 20 min and wash 3Â with 100μL wash buffer.
6. Add a final 100μL of the wash buffer to each well before the
plates are measured with the Luminex instrumentation (see
Note 8).
3.3 Sample Labeling
1. The serum or plasma samples must be thawed (see Note 9).
2. The samples are vortexed and centrifuged for 10 min at
10,000 Â g to pellet insoluble components.
3. A previously designed plate layout, in which samples should be
located randomly, is followed by transfer of 30μL of serum/
plasma into the respective wells of a PCR plate, which is then
sealed and centrifuged for 2 min at 1500 Â g.
4. Transfer 3μL into a second PCR plate containing 22μL PBS,
seal the plate, vortex, and centrifuge for 2 min at 1500 Â g.
5. Dissolve 2 mg of NHS-biotin in 200μL of DMSO and dilute it
in cold PBS to a final concentration of 4 mg/mL.
6. Add 5μL of NHS-biotin to each well (see Note 10), and then seal
the plate, vortex and centrifuge for 2 min at 1500 Â g, and
incubate for 2 h at 4
C under continuous shaking (see Note 11).
7. Add 12.5μL of cold 1 M Tris–HCl (pH 8.0) to each well. Seal
the plate, vortex, and centrifuge for 2 min at 1500 Â g.
8. Store the plates at À20
C for later analysis.
3.4 Assay Procedure
1. Thaw the labeled samples (see Note 12) and dilute them 1:10
in assay buffer supplemented with HBR6 to a final volume of
50μL. Seal the plate, vortex, and centrifuge for 2 min at
1500 Â g.
2. If desired, samples can be treated for 30 min at elevated temperatures such as 56
C in a water bath (see Note 13). Allow the
samples to cool to ambient temperature for 10 min. Vortex and
centrifuge the plate for 2 min at 1500 Â g.
3. Distribute 5μL of the bead stock solution into the wells of 96or 384-well plates and protect from light. Add 45μL of the
Minimizing Rheumatoid Factor Interference
147
immobilization efficiency through the use of fluorescently labeled
anti-species-specific antibodies.
1. Prepare the bead stock to a final concentration of 100 beads/
ID/μL.
2. Vortex the bead stock solution and sonicate for 5 min.
3. Distribute 5μL of bead mixture to each well of an assay plate.
4. Prepare the detection secondary antibody solutions described
above and add 50μL of these solutions to the wells with bead
mixture.
5. Incubate for 20 min and wash 3Â with 100μL wash buffer.
6. Add a final 100μL of the wash buffer to each well before the
plates are measured with the Luminex instrumentation (see
Note 8).
3.3 Sample Labeling
1. The serum or plasma samples must be thawed (see Note 9).
2. The samples are vortexed and centrifuged for 10 min at
10,000 Â g to pellet insoluble components.
3. A previously designed plate layout, in which samples should be
located randomly, is followed by transfer of 30μL of serum/
plasma into the respective wells of a PCR plate, which is then
sealed and centrifuged for 2 min at 1500 Â g.
4. Transfer 3μL into a second PCR plate containing 22μL PBS,
seal the plate, vortex, and centrifuge for 2 min at 1500 Â g.
5. Dissolve 2 mg of NHS-biotin in 200μL of DMSO and dilute it
in cold PBS to a final concentration of 4 mg/mL.
6. Add 5μL of NHS-biotin to each well (see Note 10), and then seal
the plate, vortex and centrifuge for 2 min at 1500 Â g, and
incubate for 2 h at 4
C under continuous shaking (see Note 11).
7. Add 12.5μL of cold 1 M Tris–HCl (pH 8.0) to each well. Seal
the plate, vortex, and centrifuge for 2 min at 1500 Â g.
8. Store the plates at À20
C for later analysis.
3.4 Assay Procedure
1. Thaw the labeled samples (see Note 12) and dilute them 1:10
in assay buffer supplemented with HBR6 to a final volume of
50μL. Seal the plate, vortex, and centrifuge for 2 min at
1500 Â g.
2. If desired, samples can be treated for 30 min at elevated temperatures such as 56
C in a water bath (see Note 13). Allow the
samples to cool to ambient temperature for 10 min. Vortex and
centrifuge the plate for 2 min at 1500 Â g.
3. Distribute 5μL of the bead stock solution into the wells of 96or 384-well plates and protect from light. Add 45μL of the
Minimizing Rheumatoid Factor Interference
147