Residues highlighted in yellow fall within the footprint recognized by PA2.1 Loxapine Succinate and BB7.2. exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1g/ml) PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50g/ml) exhibited significant cross-reactions with HLA-A*68, -A*23, and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets. Keywords:HLA class Rabbit Polyclonal to KR2_VZVD I, monoclonal antibodies, epitope, polymorphism == Introduction == Since first being reported in 19781, monoclonal antibodies with specificity for HLA class I molecules have been invaluable tools for both basic and clinical research in human immunology. These antibodies can be divided into two groups according to the types of epitope they recognize2. Monomorphic antibodies, such as W6/32, the antibody described by Barnstable et al (1), recognize monomorphic determinants that are common to all HLA class I variants, whereas polymorphic antibodies recognize determinants carried by a subset of such variants. Well-studied examples of polymorphic antibodies are PA2.1, BB7.1, BB7.2 and MA2.1. Originally, PA2.1 and BB7.2 were seen to be specific for HLA-A224, but with more extensive characterization they were also shown to recognize and define HLA-A*69, a variant that is a recombinant of HLA-A*02 and HLA-A*685. In a similar fashion, BB7.1 was originally seen to be specific for HLA-B*072but Loxapine Succinate was subsequently shown to recognize HLA-B*426, a recombinant of HLA-B*07 and HLA-B*087that is characteristic of African populations8. MA2.1, which was originally described as recognizing HLA-A2 and HLA-B17 antigens9, has been shown to react with both the B*57 and B*58 components of the HLA-B1710, but no additional reactivities have been reported. In large part, the HLA class I specificity of monoclonal antibodies has been determined using panels of cells each of which minimally expresses one HLA-A, one HLA-B and one HLA-C allotype and more commonly express two allotypes for HLA-A, -B and -C. This complexity means that binding reactions cannot be directly attributed to particular HLA class I variants but must be inferred through various types of correlation. As a consequence, there are limitations in the extent to which data can be interpreted and thus in the resolution and accuracy of the data. An initial approach to address these limitations was the use of mutant cell lines that lacked endogenous Loxapine Succinate HLA class I expression and could be transfected to express a single HLA class I allotype of choice11. A more recent approach has been to replace cells as the target antigen with synthetic beads each of which is usually coated with a single HLA class I allotype12,13. Elimination of cells from the assay facilitated the commercial development of panels of >90 different beads that provide a representation of the range of HLA-A, B and C diversity. In using such beads to determine the HLA class I specificities of Fc-fusion proteins made from killer-cell immunoglobulin-like receptors (KIR), Loxapine Succinate we have achieved results of higher resolution that are more reproducible and insightful than was possible with cell-based assays14,15. Here we have reexamined the HLA class I specificities of the MA2.1, PA2.1, BB7.2 and BB7.1 monoclonal antibodies using two panels of beads coated with.