Validation and cross-reactivity pattern assessment of monoclonal antibodies used for the screening of donor-specific IgG antibody subclasses in transplant recipients.

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 0.04 µg/mL to 5 µg/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 in IgG subclass DSA screening in allograft recipients.

Nature and Clonality of the Fluoresceinated Secondary Antibody in Luminex Multiplex Bead Assays Are Critical Factors for Reliable Monitoring of Serum HLA Antibody Levels in Patients for Donor Organ Selection, Desensitization Therapy, and Assessment of the Risk for Graft Loss.

Luminex multiplex immunoassays enable simultaneous monitoring of Abs against multiple Ags in autoimmune, inflammatory, and infectious diseases. The assays are used extensively to monitor anti-HLA Abs in transplant patients for donor organ selection, desensitization, and assessing the risk for graft rejection. To monitor IgG Abs, fluoresceinated IgG constant H chain-binding polyclonal F(ab')2 (IgHPolyFab) is used as the fluoresceinated secondary Ab (2nd-Ab), whereas IgG subclasses are monitored with Fc-specific monoclonal whole IgG (FcMonoIgG). The fluorescent signal from the 2nd-Ab is measured as mean florescence intensity (MFI). When IgHPolyFab is used, the signal is amplified as a result of the binding of multiple polyclonal Fabs to the C region of primary IgH. The reliability of such amplification for Ab measurements was not validated, nor were MFIs compared with 1:1 binding of FcMonoIgG to primary Abs. Comparing the MFIs of anti-HLA Abs obtained with IgHPolyFab and FcMonoIgG against normal human sera, IVIg, and allograft recipients' sera, it was observed that the number of HLA-Abs was notably higher with IgHPolyFab than with FcMonoIgG The MFIs of anti-HLA Abs also remained higher with IgHPolyFab in the normal sera and in IVIg, but the reverse was true when the autologous and allogeneic IgG concentrations were augmented in allograft recipients. Indeed, MFIs of the de novo allo-HLA Abs were markedly higher with FcMonoIgG than with IgHPolyFab. Serum titration established the superiority of FcMonoIgG for monitoring MFIs of de novo allo-HLA Abs in allograft recipients. Avoiding false amplifications of the number and MFIs of anti-HLA IgG with FcMonoIgG may minimize immunosuppressive therapies, maximize the number of donors for patients waiting for allografts, and enable better prediction of graft rejection.