Bio-Rad Bio-Plex Pro™ Human Cancer Biomarker Assays - Manual (Page 21)

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2. Add the required volume of Bio-Plex detection antibody diluent to a
15 ml polypropylene tube.
3. Vortex the 20x stock of detection antibodies for 15–20 sec at
medium speed, then perform a 30 sec spin to collect the entire
volume at the bottom of the tube.
4. Dilute detection antibodies to 1x by pipetting the required volume into
the 15 ml tube. Vortex.
Each well of the assay requires 1.25 μl of the 20x stock adjusted to a
final volume of 25 μl in detection antibody diluent.
Preparing 1x detection antibodies from 20x stock (includes 20% excess volume)
Table 9. Premixed panel or one singleplex assay.
Table 10. Mixing singleplex assays.
5. After incubating the beads, samples, standards, blank, and controls,
slowly remove and discard the sealing tape.
6. Wash the plate three times with 100 µl wash buffer according to the
wash method of choice.
7. Vortex the diluted (1x) detection antibodies gently for 5 sec. Pour into
a reagent reservoir and transfer 25 µl to each well of the assay plate
using a multichannel pipet.
8. Cover with a new sheet of sealing tape and incubate in the dark for
30 min at room temperature with shaking at 850 ± 50 rpm.
20x Detection Detection Antibody
# of Wells Antibodies, µl Diluent, µl Total Volume, µl
96 145 2,755 2,900
48 73 1,377 1,450
20x Detection 20x Detection Detection
Antibodies, µl Antibodies, µl Antibody
# of Wells Singleplex #1 Singleplex #2 Diluent, µl Total Volume, µl
96 145 145 2,610 2,900
48 73 73 1,304 1,450