The W6/32 monoclonal antibody recognizes a monomorphic epitope expressed on native β2 microglobulin (β2m)-associated Major Histocompatibility Complex (MHC) class I molecules: Human Leucocyte Antigen (HLA)-A, HLA-B, and HLA-C (HLA-ABC). HLA class I molecules are heterodimers comprised of an ~40-45 kDa, highly polymorphic transmembrane α heavy chain, a type I glycoprotein that is noncovalently-associated with an invariant β2-microglobulin (β2m) light chain. The N-terminal extracellular region of the HLA class I heavy chain is comprised of three domains (α1, α2, and α3). The α1 and α2 domains form a closed antigen-binding groove that accommodates 8-10 aa-peptide antigens. β2m non-covalently associates with the α3 heavy chain domain and promotes HLA class I stability. The W6/32 antibody recognizes a conformational epitope on the HLA class I heavy chain. HLA Class I antigens are normally expressed on most nucleated cells. Their expression is upregulated on activated cells or cells responding to various agents including proinflammatory cytokines or mediators. Reduced HLA Class I expression is found on certain virus-infected or tumor cells. HLA class I antigens expressed on thymic epithelial cells are involved in the positive and negative selection of CD8+ T cell precursors which determines their TCR repertoire during T cell maturation. In the periphery, these HLA Class I antigens serve to either present endogenous antigens or cross-present exogenous antigens for the generation of effector and memory CD8+ T cell responses. Target cell HLA Class I antigens can serve as ligands for inhibitory receptors expressed on NK cells and CD8+ T cells and suppress their cytotoxic responses. Human HLA class I molecules play critical roles in cell-mediated immune responses and tumor surveillance as well as tolerance to self-antigens.
The antibody was conjugated to BD Horizon BV750 which is part of the BD Horizon Brilliant™ Violet family of dyes. This dye is a tandem fluorochrome of BD Horizon BV421 with an Ex Max of 405-nm and an acceptor dye with an Em Max at 750-nm. BD Horizon Brilliant BV750 can be excited by the violet laser (405 nm) and detected with a 750/30 nm filter with a 740 nm long pass. Due to spectral differences between labeled cells and beads, using BD™ CompBeads can result in incorrect spillover values when used with BD Horizon BV750 reagents. Therefore, the use of BD CompBeads or BD CompBeads Plus to determine spillover values for these reagents is not recommended.