The L25 monoclonal antibody specifically recognizes CD49d which is also known as Integrin alpha-4 (α4 Integrin) or the alpha chain of the Very-Late Antigen-4 (VLA-4α). CD49d is a ~150 kDa type I transmembrane glycoprotein that is encoded by ITGA4 and belongs to the integrin family of cell adhesion molecules. VLA-4, like other integrins, is a noncovalently associated heterodimeric glycoprotein composed of α and β subunits and is involved in cell-cell and cell-extracellular matrix interactions. The β chain of the VLA-4 complex is the CD29 antigen, a 130 kDa glycoprotein. The CD29 antigen, also known as the β1 subunit, is common to the VLA family of integrins. When acting as a matrix receptor, the CD49d antigen binds to CS-1, an alternatively spliced domain of fibronectin. When functioning as a cell receptor, the CD49d antigen binds to the vascular cell-adhesion molecule-1 (VCAM-1). The interaction between the CD49d antigen and VCAM-1 is known to play an important role in stabilizing the adhesion of lymphocytes to endothelial cells and in mediating B-lymphocyte precursor/bone marrow stromal cell adhesion. The CD49d antigen, when associated with the β7 integrin, forms the α4/β7 integrin lymphocyte homing receptor for Peyer's patches, binding to the mucosal vascular addressin MAdCAM-1. The CD49d antigen is also involved in CD3-dependent CD4+ T-lymphocyte activation via its interaction with fibronectin. The CD49d antigen is primarily expressed on T and B lymphocytes and weakly expressed on monocytes. The L25 antibody can block or enhance fibronectin-stimulated T-lymphocyte proliferation. It immunoprecipitates three proteins of 150 kDa, 85 kDa, and 75 kDa under both reducing and nonreducing conditions from HPB-ALL cells, B lymphoblasts, peripheral blood lymphocytes, and IL-2-dependent cell lines.
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.