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HLA ANTIGEN EPITOPE DISECTION USING RECOMBINANT HLA AND A NOVEL COMPUTER ALGORITHM.
M. Bunce PhD , K. Schou C.Sci , M. Barnardo PhD , A. Mulder PhD and I. Crosby BSC . Bromborough United Kingdom, Dynal Biotech Ltd, R&D and Leiden Netherlands, LUMC, Dept Immunhaematology and Bloodtransfusion .

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Antibody screening utilizing single HLA antigens for antibody detection allows unique insights into the specificity of anti-HLA antibodies and the paratope-epitope interaction. To investigate different HLA epitopes we tested anti-HLA MoAbs with single imobilized recombinant HLA class I antigens (DynaScreen) and analysed the results using a novel algorithm (XR2) to correlate antibody reactivity to linear and conformational epitopes. An aim of this investigation was to elucidate the rules for complex paratope-epitope interactions and apply the data to construct a modified program to predict antibody crossreactivity.
XR2 analyses antibody reactivity by considering that antibody paratopes contain 1-6 contact regions (CDR) of varying size and shape, and thus the MHC epitope contact areas can also vary in size and shape. Epitope datasets used for analysis comprise contact sites that range from single residues to complex non-linear polymorphic regions with multiple residues. These contact sites are then combined in a biological model to account for the observed reactivity of an antibody, thus allowing the detection of 3-D conformational epitopes such as those combining residues on both alpha-helices. An illustration of this is the MoAb BRO11F6 that reacts strongly with HLA-A3 and A11 with weaker crossreactivity to A24 by DynaScreen. One solution that XR2 suggested for this complex specificity is a combination of the motifs TAAKR at positions 134-136,144,145 with WAA at 133,149,150 as the major epitope for A3, A11 and A24 but the absence of the spatially distant VDID motif at 76,77,95,119 for A24 results in the antibody reacting weakly with A24 and strongly with A3, A11 due to the presence of all three motifs. Other examples of the investigative power of XR2 will be given.