Chart showing the binding of two HLA-A and two HLA-B class I allotypes to the monoclonal antibody MA2.1. Results are shown relative to the pan HLA class I antibody W6/32.
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The Gen-Probe LifeCodes LSA Single Antigen Class I Bead Set offers a multiplex platform with which to test the specificity of antibodies and recombinant proteins that recognize HLA class I. The panel contains 95 HLA class I allotypes bound to individually identifiable beads. Tests are typically run on a Luminex platform that is able to read the bead identity and the fluorescence of a secondary antibody simultaneously, thus providing a readout of the level of bound antibody/recombinant protein.
Our lab uses the Gen-Probe LSA class I single antigen bead set for the determination of monoclonal antibody specificity. Monoclonal antibodies with specificity for HLA class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing three to six HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. By using a direct binding assay with HLA class I allotypes annealed to individually identifiable beads, the interpretation of the reactivity patterns is more straightforward.
Monoclonal antibodies (e.g MA2.1) against HLA class I. Antibodies should be used at a concentration of 1ug/ml.
Antibodies are incubated with Gen-Probe single antigen beads for 60min at 4°C, washed four times and then labeled with anti-mouse Fc antibody conjugated with phycoerythrin and incubated for a further 60 min at 4°C. The fluorescent intensity and identification of individual labels were visualized on a Luminex 100 reader.
The absolute amount of HLA class I annealed to each bead can be determined using the pan HLA class I antibody W6/32. Absolute binding can then be compared with the total antigen present on each bead to give relative fluorescence ratios.
Monoclonal antibodies bind with exquisite specificity to individual HLA class I allotypes. As an example, the monoclonal antibody MA2.1 bound to HLA-A*02 (and related allotypes), B*57:01 and B*58:01 consistent with it’s reported specificity of this antibody for the HLA-A2 and B17 serological antigens.
Beads should be stored for no more than 3 months at 4°C (after thawing).
Product Name: LifeCodes LSA Class I Single Antigen Bead SetSupplier Page