The 3D3 monoclonal antibody specifically recognizes FcγRII (CD32), a 40 kDa, polymorphic type I transmembrane glycoprotein that serves as a low affinity receptor for aggregated IgG. This highly glycosylated molecule (encoded by at least two different genes) is expressed on monocytes, granulocytes, platelets and B cells. Unlike the FLI28.26 mAb, the 3D3 mAb detected a polymorphic CD32 antigen expressed on B cells of all donors, but only on platelets, monocytes and granulocytes of some donors. The platelets from 3D3+ donors respond to certain stimulatory mAb such as CD165 (clone SN2) which results in aggregation. On the other hand, the platelets from 3D3 negative donors do not form aggregates after stimulation. Individuals can be divided into two groups as responder and non-responder depending on expression, or non-expression, of 3D3. In comparison to the 3D3 mAb, the FLI8.26 mAb detects a monomorphic CD32 antigen expressed on all human donors.
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.