Validation and cross-reactivity pattern assessment of monoclonal
antibodies used for the screening of donor-specific IgG antibody
subclasses in transplant recipients
Abstract
The screening for IgG subclass donor-specific antibodies (DSAs) in
allograft recipients uses IgG1-4 subclass-specific monoclonal antibodies
(mAbs) that should be mono-specific. The cross-reactivity discrepancies
reported for IgG subclass-specific mAbs warranted a critical
cross-reactivity pattern analysis of the IgG subclass-specific mAbs most
commonly used to detect DSAs. We tested the reactivity of 2 anti-IgG1-,
3 anti-IgG2-, 1 anti-IgG3-, and 2 anti-IgG4-specific PE-conjugated mAbs
against microbeads coated with IgG1-4 proteins separately. Each IgG
subclass protein was coated at three densities on the beads (0.5, 1 and
2 µg of protein per 106 beads), and the PE-conjugated mAbs were titrated
from 0.04µg/mL to 5µg/mL. The IgG subclass reactivity of the sample was
acquired on the Luminex multiplex platform. Among the IgG
subclass-specific mAbs, only the anti-IgG3 (clone: HP6050) mAb was
mono-specific. All other mAbs tested were binding to IgG subclass
proteins other than their respective immunogen, thereby being
cross-reactive. IgG subclass cross-reactivity patterns were dependent on
the concentration of both IgG subclass-specific mAbs and IgG1-4 protein
targets coated onto the beads. With the current IgG subclass mAbs
available, 3 of the 15 possible combinations of IgG1-4 subclass protein
could be identified. While the remaining 12 unique combinations cannot
be distinguished clearly, 6 groups that corresponded to two different
unique combinations of IgG1-4 subclass protein could be identified. The
dilution of serum samples and IgG subclass-specific mAbs, other than the
anti-IgG3 (clone: HP6050), must be further optimized before their
implementation of IgG subclass DSA screening in allograft recipients.