7.2
TITLE: LUMINEX TECHNOLOGY FOR TESTS OF HLA–SPECIFIC ANTIBODIES

Jeffrey T. Sholander, Julie A. Graziani, Karl P. Schillinger, Andrea A. Zachary, Mary S. Leffell.

Medicine, Johns Hopkins University, Baltimore, MD

Solid phase immunoassays using soluble HLA targets provide superior sensitivity and specificity for HLA–specific antibodies. Luminex technology uses internally stained microspheres that provide discrimination, by color, of up to 100 different beads. We have examined the LIFEMATCH™ ID (Orchid Diagnostics) (LMID), a Luminex–based assay for class I–specific antibodies. 75 sera tested with LMID were also tested by ELISA (QuikID®, GTI, Inc.) (n = 50) and/or cytotoxicity (n = 68). Compared to ELISA, LMID uses less serum volume and has a shorter run time but requires more expensive equipment for reading and analysis. LMID and ELISA had comparable ability to detect antibodies reactive in cytotoxicity but both showed increased sensitivity. However, comparing antibody specificities, the majority (23 of 37) yielded a poor correlation between LMID and both ELISA and cytotoxicity. A good correlation was obtained for only 4 antibody specificities: A2, A3, A10, and B62. Thus, antibody characterization yielded the same specificities in LMID and either ELISA or cytotoxicity only 7% of the time. In the majority of cases (83% for ELISA and 69% for cytotoxicity) there was partial overlap or inclusion of the specificities and different specficities were defined in the remaining cases. This disparity may be due to greater difficulty in defining specificity in the LMID assay. When listed in order of decreasing reactivity, antibody specificities were clustered and readily identifiable in the ELISA, but there was no obvious pattern in LMID. Further, even with limited antibodies, we saw a large range in reactivity with phenotypes bearing the target antigen. These data suggest that either there are highly variable amounts of antigen on the different beads or there is variability in the background binding that needs to be accounted for in the analysis. In summary, the Luminex technology can provide high through–put testing using small serum volumes and has the potential for testing antibodies to both class I and class II in a single tube which may compensate for the increased equipment costs. Further testing is needed to determine its accuracy in characterizing antibodies.