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™ BUV563 which is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This dye is a tandem fluorochrome of BD Horizon BUV395 which has an Ex Max of 348 nm and an acceptor dye. The tandem has an Em Max at 563 nm. BD Horizon BUV563 can be excited by the 355 nm ultraviolet laser. On instruments with a 561 nm Yellow-Green laser, the recommended bandpass filter is 585/15 nm with a 535 nm long pass to minimize laser light leakage. When BD Horizon BUV563 is used with an instrument that does not have a 561 nm laser, a 560/40 nm filter with a 535 nm long pass may be more optimal. Due to the excitation and emission characteristics of the acceptor dye, there may be spillover into the PE and PE-CF594 detectors. However, the spillover can be corrected through compensation as with any other dye combination.