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™ 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.