EVALUATION OF AN ELISA ASSAY USING SYNTHETIC A AND B CARBOHYDRATES TO ASSESS ANTIBODY LEVELS IN ABO INCOMPATIBLE KIDNEY TRANSPLANT PATIENTS

Abstract

A282 While the outcomes of ABO incompatible kidney transplantation generally correlate with the baseline antibody level as determined by hemagglutination, we have observed several occurrences when patients with low hemagglutination titers have developed humoral rejection. We hypothesized that the hemagglutination assay (HA) may underestimate the level of anti-AB antibodies and may provide targets for antibodies other than A and B. The Aim of this study was to develop a specific ELISA assay for anti-AB antibodies using synthetic A and B carbohydrates and to compare the results with HA. Methods: ELISA plates with low (LB) or high (HB) protein binding capacity were coated with polyacril-amid (PAA)-A or B antigens. Uncoated wells, wells coated with PAA, and with PAA-irrelevant trisacharide (PAA-Ir) served as controls for nonspecific binding (NSB). Human serum samples were applied in 2 fold dilutions followed by incubations with monoclonal antibodies against IgM, IgG, IgA classes/subclasses, anti-mouse-peroxidase antibody and ABTS substrate. The positive result was determined as serum dilution (titer) after the NSB of the controls was deducted. HA was performed as immediate spin (IS) agglutination and anti-human globulin (AHG) test. Results: The LB plates showed low NSB but also low specific binding, which accounted for false negative results. The HB plates showed higher background, which was similar in the uncoated wells, PAA wells and PAA-Ir wells which demonstrates that the NSB is caused by absorption of proteins on the plastic. The NSB was corrected by using the intra assay coefficient of variation (IACV) based on the formula ODAB –2IACV>ODContr+2IACV. Interassay CV for anti-AB IgM/G/A was from 4.6 to 9.7%. The specificity of the assay was confirmed by negative results with PAA-Ir. The sensitivity of the ELISA assay was 2.77 two-fold dilutions higher than the HA (p<0.0001). IgM ELISA titers correlated with the IS results (p < 0.001). No correlation was found between IgG ELISA titers and AHG. Titers for IgG2 were generally higher than IgG1. Anti-AB IgA was found mostly in blood type O. We examined the ELISA levels before and after plasmapheresis in 5 patients. ELISA showed measurable anti-AB antibody levels after plasmapheresis when the HA titers were zero. The levels of IgM anti-AB antibodies after plasmapheresis in 2 patients were ≥ 128; one of these patients later developed antibody mediated rejection and the other developed chronic allograft nephropathy. Conclusions: 1) The sensitivity of ELISA was significantly higher than HA. 2) IgM anti-AB antibody levels showed correlation with IS while IgG did not show correlation with AHG, which might be due to residual IgM antibodies participating in AHG agglutination. 3) Preliminary data suggest that ELISA IgM anti-AB levels after plasmapheresis when HA levels are not measurable may predict negative outcome. Corollary: These data supported that the ELISA assay is sensitive and specific and would allow for better evaluation of the role of anti-AB antibodies in ABO incompatible grafts.