P045 Complement binding to single antigen beads is not directly related to antibody strength as determined in pairwise comparative studies

Abstract

Aim Optical bead microarrays using Luminex have largely replaced anti-HLA antibody screening tests using cell based complement dependent cytotoxicity assays. One unintended consequence of this technologic development was the loss of the ability to distinguish between antibodies that fix complement from those that do not. This situation may have been addressed by the introduction of several assays that use the bead microarrays in conjunction with a reagent that detects whether components of the complement cascade are bound to the anti-HLA antibody – HLA complex on a reactive bead. One controversy centering on this technology has been the suggestion that the binding of complement in bead assays is dependent to a greater extend on the strength of the anti-HLA antibody rather than its complement binding activity. To address this issue we examined complement binding in single antigen bead (SAB) assays in sera that had multiple epitope specificities of different strengths. Methods Patient sera and sera from proficiency surveys were used. SAB assays of these sera were compared to C1q/C3d – SAB assays. The bead with the highest net MFI for each identified epitope in the microarray was recorded and compared to the same bead in C1q/C3d – SAB assays. Results We detected both strong and weak anti-HLA antibodies in SAB assay that were detected in C1q/C3d – SAB assays. In some sera complement bound to one set of epitope reactive SABs but not to another epitope set of SABs. Importantly, this was not dependent on the strength of the antibody. For example, in one patient’s sera, an epitope defined antigen bead, B∗42:01 (epitope 65GK), had an MFI of 12,989 (normalized score) in the SAB assay but only 99 MFI in the C1q – SAB assay. In the same serum another epitope defined bead (80 N), A∗23:01 with an MFI of 10,445 in the SAB assay exhibited an MFI of 9,162 in the C1q – SAB assay. We also observed the latter finding for HLA class 2 SABs and with the C3d assay. Conclusions Complement binding to SABs is not directly related to strength of the antibody as determined in pairwise comparative studies using sera that contained antibodies with different epitope specificities. C1q/C3d - SAB assays are therefore useful in evaluating the complement binding properties of pretransplant and post transplant anti-HLA antibodies.