This kit is designed for the detection of binding between FCGR3B (CD16b) and human IgG Fc fragment using a homogeneous AlphaLISA assay (no wash steps). This assay can facilitate the design and development of antibody therapeutics by using a competition assay format.

AlphaLISA features:

• No-wash steps, no separation steps
• Ease-of-use: few addition steps, fast assay development
• Broad range of affinities: detect strong or weak interactions, from pM to mM affinity
• Distance: measure very large protein or antibody complexes – spanning up to 200 nm or more
• High avidity: multiple binding sites on each bead enables use of nanomolar concentrations of antibodies or proteins, as well as use of low affinity binders

The Fc-Gamma Receptors (FCGRs) are members of immunoglobulin superfamily and play a critical role in the function of therapeutic antibodies. FCGRs are divided into three classes Fc-Gamma Receptor 1 (CD64), FCGR1; Fc-Gamma Receptor 2 (CD32), FCGR2 and Fc-Gamma receptor 3 (CD16), FCGR3. FCGR3 is expressed as two distinct forms (FCGR3A and FCGR3B) encoded by two different highly homologous genes in a cell type specific manner. FCGR3 is a low/intermediate affinity receptor for polyvalent immune-complexed IgG. FcGR3B binds complexed or aggregated IgG and also monomeric IgG. Contrary to FCGR3A, FCGR3B is not capable to mediate antibody-dependent cytotoxicity and phagocytosis. It may serve as a trap for immune complexes in the peripheral circulation which does not activate neutrophils.

AlphaLISA detection of FCGR3B and IgG Fc fragment binding uses IgG Fc AlphaLISA® Acceptor beads and Streptavidin-coated Donor beads to capture biotinylated human FCGR3B. Donor beads and Acceptor beads come into proximity through IgG Fc fragment binding to FCGR3B. Excitation of the Donor beads provokes the release of singlet oxygen that triggers a cascade of energy transfer reactions in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm.