Detection and specification of noncomplement binding anti-HLA alloantibodies.

The aim of the study was to investigate the distribution of human leukocyte antigen (HLA) -specific immunoglobulin (Ig) isotypes/subclasses in alloimmunized patients awaiting a kidney retransplant. Sera from 102 patients were analyzed for the presence of anti-HLA-A, anti-HLA-B alloantibodies by complement-dependent cytotoxicity test with the addition of dithiothreitol (CDC+DTT). Furthermore, anti-HLA class I and class II alloantibodies were determined using a commercial solid-phase (enzyme-linked immunosorbent assay [ELISA]) system. The respective isotypes/subclasses were defined by replacing the IgG1-4 secondary antibody with IgG1-, IgG2-, IgG3-, IgG4-, IgA1-, IgA2-, and IgM-specific antibodies. The HLA specificities of the noncomplement-binding IgG2 and IgG4 antibodies were determined and compared with the mismatches from the failed transplants. Thirty-eight of 102 (37%) sera were positive in the class I CDC+DTT, in contrast to 41 of 102 (40%) detected by class I ELISA and 47 of 102 (46%) by class II ELISA. Seventeen of 102 (17%) positive reaction were observed for the IgM-isotype, whereas none were detected for the IgA-isotype. Twenty-five of 102 (25 %) sera contained noncomplement-binding IgG2 and/or IgG4 antibodies; in the majority of the cases, 22 of 25 (88%) were directed against the organ donor antigen. These data show that donor-specific, noncomplement-binding IgG2 and IgG4 alloantibodies exist with high prevalence in HLA-immunized retransplant candidates. Therefore, a thorough antibody screening workup, including CDC with or without DTT and ELISA screening should be performed for patients before they reenter the waiting list. Defining the Ig isotypes and subclasses can be helpful to explain inconsistent results.