The 2H9 monoclonal antibody specifically binds to the Ephrin Type-B Receptor 2 (EphB2). EphB2 is a type I transmembrane glycoprotein that belongs to the Eph receptor family of tyrosine kinase receptors. EphB2 serves as a cell surface receptor tyrosine kinase for membrane-anchored ligands referred to as type B ephrins (ephrin-B). The EphB2 receptor can bind to ephrin-B1, ephrin-B2, and ephrin-B3. Transmembrane ephrin-B family members are key regulators of embryogenesis including development of the nervous and vascular systems. The EphB2 receptor functions as a chemodirectant in regulating cellular migration. EphB2/ephrin-B interactions orchestrate cell positioning by regulating cellular adhesion and repulsion during development, thereby influencing cell fate, morphogenesis and organogenesis. Signaling can occur in a forward pathway when the EphB2 receptor tyrosine kinase is activated by bound ligand and in a reverse pathway when transmembrane ephrin-B ligands are activated by EphB2 receptor-mediated crosslinking. In the adult body, Eph receptor signaling plays major roles in regulating the architecture and physiology of different tissues under normal as well as disease conditions such as cancer. Ephrin-B1 and ephrin-B2 levels are upregulated in the vasculature during inflammation. Ephrin-B2 molecules that are localized to the luminal endothelial surface can signal through the EphB2 which is expressed by monocytes. This interaction promotes monocyte differentiation into proinflammatory macrophages. In the intestinal epithelium, EphB2/ephrin-B interactions regulate both cell positioning and tumor progression. The differential expression patterns of EphB2 allows for the detection and isolation of various intestinal epithelial cell types. These include intestinal stem cells (ISCs) which express high levels of EphB2. The 2H9 antibody reportedly blocks the interaction of EphB2 with ephrin ligands and crossreacts with mouse EphB2.
The antibody was conjugated to BD Horizon™ BUV661 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 661-nm. BD Horizon Brilliant BUV661 can be excited by the ultraviolet laser (355 nm) and detected with a 670/25 filter and a 630 nm LP. Due to cross laser excitation of this dye, there may be significant spillover into channels detecting APC-like emissions (eg, 670/25-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 BUV661 reagents. Therefore, the use of BD CompBeads or BD CompBeads Plus to determine spillover values for these reagents is not recommended. Different BUV661 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.