The 2A3 monoclonal antibody specifically binds to human CD25, the low-affinity alpha subunit of the Interleukin-2 Receptor (IL- 2Rα). CD25 associates with CD122 (IL-2Rβ chain) and CD132 (common γ chain or γc) to form the high-affinity signal-transducing IL-2R complex. CD25 is expressed by subsets of thymocytes and peripheral blood lymphocytes including CD4+CD25+ regulatory T cells and memory T cells. CD25 antigen density increases on activated T cells including phytohemagglutinin (PHA)-, concanavalin A (Con A)-, and CD3-activated T lymphocytes. High levels of CD25 can be expressed by T lymphocytes from mixed lymphocyte cultures and by human T-lymphocyte leukemia virus (HTLV)-infected T-lymphocyte leukemia lines, for example, HUT-102. CD25 can also be expressed by activated B cells and macrophages. Recombinant IL-2 blocks the binding of the 2A3 antibody to PHA-activated T lymphocytes.
The antibody was conjugated to BD Horizon™ BUV496 which is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This dye is a tandem fluorochrome of BD Horizon BUV395 with an Ex Max of 348-nm and an acceptor dye with an Em Max at 496-nm. BD Horizon BUV496 can be excited by the ultraviolet laser (355 nm) and detected with a 515/30 nm filter with a 450LP. Due to the excitation of the acceptor dye by other laser lines, there may be significant spillover into the channel detecting BD Horizon V500 or BV510 (eg, 525/40-nm filter). However, the spillover can be corrected through compensation as with any other dye combination.