Introduction
Since the publication of “Humoral Theory of Transplantation” by Terasaki PI (1), the close association between donor-specific antibodies (DSA) and antibody-mediated rejection (ABMR) has been widely established across all organs, and has become accepted as a major cause for poor long-term allograft outcomes (2–7). In addition to allograft recipients transplanted in the presence of preformed DSA, roughly 20% of allograft recipients develop de novo DSA post-transplantation (8), and the presence of DSA has been implicated in a wide range of histopathological findings from the absence of antibody-mediated injuries, to indolent subclinical ABMR, to acute ABMR, and to hyperacute rejection (9). Consequently, it has been realized that not all DSAs are equally pathogenic, particularly because, unequivocally, about 20-50% of allograft recipients with DSA do not experience worse allograft outcomes compared to patients without DSA (8). This observation led to the evaluation of the impact of different functional characteristics of DSAs, related to the effector functions of IgG1-4 subclasses, on antibody-mediated injuries and allograft outcomes (8,10,11).
The indirect effector functions of IgG DSAs – largely governed by their subclass – are mediated by the Fc region engagement, and include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) (12). For example, while IgG3 and IgG1 are potent complement activators, IgG2 poorly activates complement and IgG4 does not fix complement. Although the heavy chain constant regions are more than 90% homologous across IgG subclasses, important variations within the hinge region, the CH2 domain and the CH3 domain affects the binding affinities to Fcγ receptors (FcγR), complement C1q, and neonatal Fc receptor (FCRn) of each IgG subclass (12). In addition, the molecular differences within the Fc region of IgG1-4 subclasses can also serve as monoclonal antibody epitopes. Interestingly, the generation of monoclonal antibodies (mAbs) raised against purified IgG1-4 proteins, separately, documented that some mAbs were binding to their immunogen only (mono-specific) while others were binding to more than one IgG subclass protein (cross-reactive) (13). Certainly, the detection of IgG subclass-specific DSAs should be performed using mono-specific anti-IgG subclass mAbs and the cross-reactive ones should be avoided.
The development of solid-phase assays using the Luminex® multiplex platform greatly improved the specificity and sensitivity of DSA screening, and enabled the categorization of DSA based on their functional characteristics, namely C1q-binding and IgG1-4 subclasses. The Luminex® platform uses antigen-coated beads to detect serum antibodies of interest and requires a phycoerythrin (PE)-conjugated secondary antibody. For the detection of DSA IgG subclasses, several PE-conjugated mAbs – most of which were generated by Reimer C et al. in 1984 (13) – are commonly used, yet procedures/validation methods are not standardized across HLA laboratories. Nonetheless, an apparent consensus on the mono-specificity of the IgG subclass-specific PE-conjugated mAbs was reached provided the mAbs were used at a concentration that minimized cross-reactivity (<5-15%) (14–16); yet one study has reported a 37% IgG4 cross-reactivity for an anti-IgG1 specific mAb (17). Recently, the PE-conjugated secondary antibody used to detect DSA has been shown to be an important factor for the determination of DSA strength, expressed as mean fluorescence intensity (MFI), where not only the concentration of the PE-conjugated secondary antibody affects MFI output but also the concentration of the primary antibody bound to the antigen-coated beads (18).
The minimal discrepancies in the cross-reactivity pattern reported for IgG subclass-specific mAbs and the absence of detailed technical descriptions of the validation assays warranted a critical cross-reactivity pattern analysis of IgG subclass-specific mAbs used for the detection of DSAs in current and prospective transplant recipients. Therefore, the goal of this study is to evaluate the cross-reactivity pattern of several IgG subclass-specific mAbs using the Luminex® multiplex platform with different beads covalently coated with purified human IgG1, IgG2, IgG3, or IgG4 proteins, and following a box-titration for both IgG subclass-specific mAbs and for IgG1-4 protein targets.