P058 Validation of anti-human antibodies used in flow cytometric crossmatching

Abstract

Aim Flow cytometric crossmatches (FCXM) are widely used to detect recipient alloantibodies against donor tissue to assess compatibility for transplantation. Polyclonal anti-human globulin (AHG) is the reagent typically utilized for the detection of these alloantibodies. Humans produce four primary subtypes of IgG, designated as IgG1–4. Here, we describe a system to determine whether the AHG is able to detect all IgG subtypes. Methods Humanized monoclonal antibodies of each IgG subtype, specific for a red blood cell (RBC) antigen were produced. Briefly, the cDNA for the complimentarity-determining regions (CDR) of the heavy and light chains specific for the Kell RBC antigen were ligated into expression vectors for the separate IgG subtypes 1–4 and kappa light chains, co-transfected into CHO cells, and purified. These monoclonal IgG1–4 antibodies were then tested against RBCs, and three different lots of AHG were used to detect the antibodies bound to the RBCs in a standard FCXM assay. An anti-kappa light chain antibody that recognizes an epitope common in all IgG subtypes was used as a positive control. The AHG alone was used as a baseline control. Results As shown in Fig. 1, each lot of AHG was able to detect all four subtypes of IgG. In addition, titration experiments were performed to determine whether there was preferential detection of any IgG subtype(s). These experiments demonstrated that AHG does exhibit preferential binding to different IgG subtypes. In one specific lot of AHG, the hierarchy of binding/detection was IgG3 > IgG1 > IgG2 > IgG4. Download : Download high-res image (264KB) Download : Download full-size image Conclusion These data provide a proof-of-principle for a method to characterize and validate AHG reagents used in FCXM assays. This is important, because the inability of AHG reagents to detect IgG subtypes may lead to false-negative FCXM results. These animal-derived AHG reagents are polyclonal, and there may be lot-to-lot variability in their ability to detect different IgG subtypes. J. Zimring: Grant/Research Support; Company/Organization; Immucor, Inc.. Scientific/Medical Advisor; Company/Organization; Rubius Therapeutics. Employee; Company/Organization; Bloodworks NW.