The monoclonal antibodies TH6 and 12A5 recognize Folate Receptor 4 (FR4), also known as the membrane folate-binding protein 3 (FBP3). FR4 is a heavily glycosylated 35 kD receptor expressed exclusively in lymphoid tissue and an isoform of the family of receptors that recognize the essential nutrient folic acid. Natural T regulatory cells constitutively express high levels of FR4. Differential expression of FR4 in combination with CD25 can distinguish four functionally distinct CD4+ T cell subpopulations; Natural Tregs, effector T cells, memory-like T cells and Naïve T cells. FR4hi CD25+ expressing CD4+ T cells also express high amounts of Foxp3, GITR and CTLA-4.
Monoclonal antibody TH6 and 12A5 stained CD25+CD4+ T cells at a higher level than other CD4+ or CD8+ T cells. In addition, in vivo injection of TH6 monoclonal antibody reduced the number of CD25+CD4+ T cells and CD25-CD4+ T cells in peripheral blood. Clone 12A5 has been demonstrated to work in western blot. Clones TH6 and 12A5 do not block one another in a flow cytometric assay.
The antibody was conjugated to BD Horizon BUV615 which is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This dye is a tandem fluorochrome with an Ex Max near 350 nm and an Em Max near 615 nm. BD Horizon Brilliant BUV615 can be excited by the ultraviolet laser (355 nm) and detected with a 610/20 filter and a 595 nm LP. Due to the excitation of the acceptor dye by the blue/yellow-green laser line, there may be significant spillover into channels detecting PE-CF594 like emissions (eg, 610/20-nm filter).