Characterization of anti-HLA antibodies eluted from explanted renal allografts

To evaluate the immunoglobulin isotypes of anti-HLA antibodies harbored in rejected renal allografts, we isolated proteins by acid elution accumulated in 94 rejected and explanted kidneys and characterized their antibody specificities by CDC, ELISA, and flow cytometry (Luminex) techniques. In addition, we differentially analyzed non-complement binding IgG2/4 and IgA1/2 antibodies in the eluates using two modified solid phase assays. The comparative analysis of anti-HLA class I and II antibodies with donor specificity (ELISA vs. Luminex) revealed 30% vs. 33% class I IgGall positive eluates, whereas 26% vs. 15% of the eluates had donor-specific anti-HLA class II IgGaII antibodies. Regarding the non-complement binding antibodies we could show IgG2 and IgG4 antibodies in 16/58 (28%) of the IgGaII positive eluates: 15 eluates with anti-HLA class I and 4 with anti-HLA class II specificities, respectively. Anti-HLA class I IgG2/4 antibodies directed against the donor were found in 7 eluates (54% of the IgG2/4 pos. eluates), whereas 2 eluates (50%) had class II IgG2/4 antibodies directed against the donor. IgA1/2 antibodies could be detected in 9 eluates (16%), 5 of them had anti-HLA class I and 5 anti-HLA class II antibodies. We could clearly show that explanted kidney allografts harbor anti-HLA antibodies, when sensitive antibody detection techniques are used. Moreover, our study demonstrates that non-complement binding anti-HLA antibodies also accumulate in renal allografts.