Monoclonal secondary reagents do not outperform polyclonal secondary reagents for detection of anti-HLA IgG using single antigen flow beads assays.

Abstract

Luminex single antigen flow beads (SAFB) assays are used to monitor anti-HLA antibodies, which are associated with allograft loss. The primary antibody binding level is evaluated by the mean fluorescence intensity (MFI) provided by the tracing secondary antibody. The aim of this study was to compare the MFI obtained with two secondary antibodies and to evaluate their sensitivity to detect newly developed de novo anti-HLA donor-specific antibodies (dnDSA). A total of 23 and 46 different sera from 20 HLA-sensitized kidney transplant recipients were tested with class I and II SAFB, respectively, as well as sera from 17 patients with dnDSA. The conventional secondary antibody (IgHPolyFab) was the phycoerythrin (PE)-conjugated goat anti-human IgG constant heavy chain (HC)-binding polyclonal-F(ab')2. The alternate secondary antibody was a PE-conjugated anti-human IgG Fc-specific monoclonal IgG (FcMonoIgG). MFI of negative control sera were drastically lower with the FcMonoIgG than with IgHPolyFab, requesting a unique positivity threshold to be defined for each secondary antibody. MFI obtained with positive control sera were also lower with FcMonoIgG. In kidney transplant recipient's sera, the number of antigenic specificities detected by each secondary antibody was comparable, for dnDSA as well as for nonDSA. In conclusion, decision thresholds must be refined when changing the secondary tracing reagent in SAFB assays. FcMonoIgG did not demonstrate any superiority over IgHPolyFab for monitoring HLA antibodies.