AMINO ACID RESIDUES DETERMINING EPITOPES ON HLA-A ALLELES RECOGNIZED BY MONOCLONAL ANTIBODIES IN FLOW CYTOMETRY.

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AMINO ACID RESIDUES DETERMINING EPITOPES ON HLA-A ALLELES RECOGNIZED BY MONOCLONAL ANTIBODIES IN FLOW CYTOMETRY.
Hugo A. Araujo , Martin Maiers , Martin Gutierrez , Arend Mulder , Jar Haw Lee , Jennifer Ng , Robert J. Hartzman and Marcelo A. Fernandez-Vina . Kensington MD, Naval Medical Research Center/Georgetown University, C.W.Bill Young DoD Marrow Donor Program ; Minneapolis MI, National Marrow Donor Program, Information Systems ; Leiden Netherlands, Leiden University Medical Center ; Canoga Park CA, One Lambda, Inc. and Washington DC, Georgetown University, Department of Oncology, Lombardi Cancer Center .

Several strategies were developed to determine the correlation between typing reagents and HLA specificities. The 13^th IHWC incorporated an additional mapping strategy to predict HLA specificities for untested alleles. Still, mapping the amino acid residues with monoclonal antibodies (mAb) by flow cytometry is a crucial experimental tool to assess the validity of such predictions. Twenty-one HLA-A monoclonal antibodies (mAb) were labeled with Alexa 488 and assayed with 69 BLCL by flow cytometry. Results were defined by fluorescence intensity measured as median channel values (MCV). Cut-off limits to distinguish positive and negative reactions for each BLCL were calculated as the median + 2 SD of all expected negative reactions. We found 4 mAb clustering A*23 and A*24 with amino acids D166 and G177; the additional activity for A*01 and A*8001 further included N77 and/or E62. Amino acid replacement of DG by EW on residues 166 and 167, respectively, caused loss of reactivity for A*2304, A*2403 and A*2422 with mAb 13^th IHWC 0004, 0006, 0007 and 0047. Residues D161 could be part of an epitope carried by A*0301/0302 molecules and recognized by mAb 13^th IHWC 0033 and 0038. Another A3-related epitope includes Q70 and/or I97 which clusters A3 with A11, A30, A31, A33 and perhaps A29 detected by 13^th IHWC 0040, 0041, 0043 and 0060. Direct binding with HLA mAb labeled with Alexa 488 assayed by flow cytometry refined the clustering of HLA-A alleles according to amino acid residues conforming epitopes. Precise epitope mapping is crucial to assess cell surface expression of HLA products.