Bio-Rad Bio-Plex Pro™ Human Isotyping Assays - Manual (Page 16)

14
6. Prepare Samples
These assays are designed to quantitate classes and subclasses of
immunoglobulins in serum, plasma, and cell culture media. For optimal
recovery and sensitivity, it is important to properly prepare samples.
n
once thawed, keep samples on ice. Prepare dilutions just prior to the
start of the assay and equilibrate to room temperature before use
n
Prepare sample dilutions in 1.5 or 2 ml microcentrifuge tubes. If a
multichannel pipet will be used to load the plate, then aliquot the required
volumes into Titertube
® micro test tubes.
n
Do not freeze diluted samples
Sample Preparation
Serum and Plasma
EDTA or citrate is preferred as an anticoagulant. Heparin-treated plasma,
while compatible with Bio-Plex Pro
™ assays, may absorb certain soluble
proteins of interest. Avoid using hemolyzed samples as this may lead to
false positive results.
1. Draw whole blood into collection tubes containing anticoagulant.
Invert tubes several times to mix.
2. For serum, allow blood to clot at room temperature for 30 to 45 min.
For plasma, proceed directly to the centrifugation steps.
3. Perform centrifugation at 1,000 x g for 15 min at 4°C and transfer the
serum or plasma to a clean polypropylene tube.
Table 5. Summary of recommended sample diluents and dilution factors.
Sample Type
Diluent Add BSA Recommended Sample Dilution
Serum and plasma Isotyping None 1:40,000 for IgG
1
, IgG
2
, IgG
3
, IgG
4
,
diluent IgA, IgM, IgG total
1:500 for IgE
Culture media, with serum Culture media None User defined
Culture media, serum-free Culture media To 0.5% final User defined