The 232-5 monoclonal antibody specifically recognizes Butyrophilin 3 (BTN3), which is also known as CD277. BTN3 is comprised of three structurally related members, BTN3A1, BTN3A2, and BTN3A3. BTN3 molecules are transmembrane type 1 glycoproteins that belong to the Ig superfamily. The 232-5 antibody recognizes the variable region of the extracellular domains of BTN3 molecules. BTN3 molecules are differentially expressed by T, NKT, B, and NK cells, monocytes, macrophages, dendritic cells, as well as some stem cells and tumor cells. BTN3 expression is decreased upon lymphocyte activation. The extracellular domain of CD277 is structurally related to the B7 family of proteins that regulate T cell activation. A soluble form of the 232-5 antibody can serve as an agonist in BTN3-mediated regulation of anti-CD3 ± anti-CD28 antibody activated CD4+ or CD8+ T cells.
The antibody was conjugated to BD Horizon™ BUV737 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 737-nm. BD Horizon Brilliant BUV737 can be excited by the ultraviolet laser (355 nm) and detected with a 740/35 filter. Due to the excitation of the acceptor dye by other laser lines, there may be significant spillover into channels detecting Alexa Fluor® 700-like dyes (eg, 712/20-nm filter).
Due to spectral differences between labeled cells and beads, using BD™ CompBeads can result in incorrect spillover values when used with BD Horizon BUV737 reagents. Therefore, the use of BD CompBeads or BD CompBeads Plus to determine spillover values for these reagents is not recommended. Different BUV737 reagents (eg, CD4 vs. CD45) can have slightly different fluorescence spillover therefore, it may also be necessary to use clone specific compensation controls when using these reagents.