Bio-Rad Bio-Plex Pro™ RBM Canine Kidney Toxicity Assays - Manual (Page 2)

B. Dilution of Standard (1:3 Serial Dilution)
1. Label 8 polypropylene tubes S1 through S8.
2. Transfer the reconstituted standard into the tube labeled “S1.”
3. Add the appropriate amount of the standard diluent into the labeled
tubes according to the table below (this will be sufficient for duplicate
standard curves and blanks).
4. Prepare working standards (S2–S8) by serial dilution. Transfer the
appropriate volume of standard into each of the labeled tubes with
standard diluent as outlined above.
5. Vortex each standard at a medium setting before proceeding with the next
serial dilution. Change pipet tip at each dilution step.
C. Sample Preparation
1. Centrifuge samples at 500 x g for 5 min to remove particulates from all
samples prior to use.
2. Prepare sample dilutions in 0.5 ml or 1.0 ml polypropylene tubes as
required for the assay.
3. Dilution scenarios provided below are sufficient to run each sample
in duplicate.
Bio-Plex Pro Assay Quick Guide Bio-Plex Pro Assay Quick Guide
Standard Volume of Standard Diluent, µl Volume of Standard, µl
S2 100 50 of S1
S3 100 50 of S2
S4 100 50 of S3
S5 100 50 of S4
S6 100 50 of S5
S7 100 50 of S6
S8 100 50 of S7
Blank 100 —
D. Dispensing of Reagents
1. Add 10 µl of blocker to all wells of the plate.
2. Add 30 µl of the standard, control, sample, or blank to the appropriate well
of the plate.
3. Vortex the capture beads at medium speed for 10–20 sec. Add 10 µl of the
beads to all wells of the plate.
4. Cover plate with plate seal and protect from light with aluminum foil.
Incubate on shaker at 850 ± 50 rpm for 1 hr at RT.
5. Wash the plate three times with 100 µl 1x assay buffer.
6. Vortex the reconstituted detection antibodies at medium speed for
10–20 sec. Add 40 µl to each well.
7. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 1 hr at RT. Do not
aspirate after incubation.
8. Prepare the required dilution of SA-PE as outlined in the following table.
Note: Volumes in the table are for an entire 96-well plate. Smaller volumes
can be prepared, provided that dilution ratios are maintained.
9. Add 20 µl of diluted SA-PE to the required plate wells.
Volume of Volume of
Panel Sample Dilution Urine Sample, µl Sample Buffer, µl
Human Tox 1 1:4 20 60
Human Tox 2 1:50 10 490
Rat Tox 1 1:2 40 40
Rat Tox 2 1:50 10 490
Rat Albumin 1:10,000 5 (A. Prepare 1:100) 495
5 (B. Prepare 1:100) 495
Canine Tox 1 1:15 10 140
Canine Albumin 1:10,000 5 (C. Prepare 1:100) 495
5 (D. Prepare 1:100) 495
Note: Controls are ready to use after reconstitution. No dilution is needed.