Removal of cells from your 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. cross-reactivity with HLA-A*11 and -B*15:16. At low Carboxin 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 acquired with Rabbit Polyclonal to SEPT7 cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes identified by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Assessment of two overlapping but special bead units 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 units. strong class=”kwd-title” Keywords: HLA class I, monoclonal antibodies, epitope, polymorphism Intro Since first becoming reported in 19781, monoclonal antibodies with specificity for HLA class I molecules have been priceless tools for both fundamental and clinical study in human being immunology. These antibodies can be divided into two organizations according to the types of epitope they identify2. Monomorphic antibodies, such Carboxin as W6/32, the antibody explained by Barnstable et al (1), identify monomorphic Carboxin determinants that are common to all HLA class I variants, whereas polymorphic antibodies identify 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-A22C4, 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*072 but was subsequently shown to recognize HLA-B*426, a recombinant of HLA-B*07 and HLA-B*087 that 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 communicate 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 degree 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 HLA class I expression and could be transfected to express a single HLA class I allotype of choice11. A more recent approach offers been to replace cells as the prospective antigen with synthetic beads each of which is definitely coated with a single HLA class I allotype12,13. Removal of cells from your 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), we have accomplished 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 Carboxin MA2.1, PA2.1, BB7.2 and BB7.1 monoclonal antibodies using two panels of beads coated with HLA class I molecules. Materials and Methods Binding of MA2.1, PA2.1, BB7.2 and BB7.1 antibodies to beads, each coated having a representative range of HLA-A, HLA-B and HLA-C allotypes was assessed inside a multiplex assay within the Luminex platform (Austin, TX). The bead panels tested were (a) LabScreen single-antigen beads (One Lambda, Canoga Park, CA) and (b) LifeCodes single-antigen beads (Gen-Probe, San Diego, CA). Antibodies (1g/ml and 50g/ml) were incubated with each set of beads for 60 mins at 4C, washed four times and then labeled with anti-mouse Fc antibody conjugated with phycoerythrin and incubated for a further 60 mins at 4C..