IgG subclass validation assay
The IgG subclass-specific beads were used to evaluate the reactivity
pattern of the anti-human-IgG subclass-specific mAbs listed in Table 1.
For each concentration of coating (0.5μg, 1μg, or 2μg per
1x106 beads), equal volume and number of IgG1-, IgG2-,
IgG3-, and IgG4-coated beads and a negative control bead (coated with
bovin serum albumin) were combined to a final mixture concentration of
2000 beads per μL, and then tested in a multiplex fashion. Briefly, 2μL
of IgG subclass beads mixture was mixed with 20μL of 1X PBS for 30
minutes in the dark at room temperature. The beads were then washed
three times with 180μL of 1X wash buffer (One Lambda Inc, Canoga Park,
CA). Next, we added 50μL of PE-conjugated anti-human-IgG
subclass-specific mAbs samples. All anti-human-IgG subclass-specific
mAbs were titrated (5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08, and 0.04
μg/mL) and tested separately against beads coated with 0.5μg, 1μg, and
2μg per 1x106 beads of IgG subclass protein. Then, IgG
subclass-specific beads were incubated for 30 minutes in the dark at
room temperature. Again, the beads were washed three times with 180μL of
1X wash buffer, and 60μL of 1X PBS was added before the data was
acquired on a Luminex 100 analyzer. At least 100 beads were counted for
each IgG subclass-specific bead and for each sample. The reactivities
against each IgG subclass protein were recorded as Trimmed Mean
Fluorescence Intensity (MFI). All MFI values reported were normalized
against the negative control bead.