Anti-Mouse CD16.2 (Clone 9E9) – Purified in vivo PLATINUM™ Functional Grade

This monoclonal antibody is aseptically packaged and formulated in 0.01 M phosphate buffered saline (150 mM NaCl) PBS pH 7.2 - 7.4 with no carrier protein, potassium, calcium or preservatives added. Due to inherent biochemical properties of antibodies, certain products may be prone to precipitation over time. Precipitation may be removed by aseptic centrifugation and/or filtration.

Functional grade preclinical antibodies are manufactured in an animal free facility using in vitro cell culture techniques and are purified by a multi-step process including the use of protein A or G to assure extremely low levels of endotoxins, leachable protein A or aggregates.

To protect mouse colonies from infection by pathogens and to assure that experimental preclinical data is not affected by such pathogens, all of Leinco’s Purified Functional PLATINUM^TM antibodies are tested and guaranteed to be negative for all pathogens in the IDEXX IMPACT I Mouse Profile.

Functional grade preclinical antibodies may be stored sterile as received at 2-8°C for up to one month. For longer term storage, aseptically aliquot in working volumes without diluting and store at -80°C. Avoid Repeated Freeze Thaw Cycles.

Next Day 2-8°C

9E9 activity is primarily directed against mouse CD16.2 / FcγRIV but can also bind and block FcγRIII in vivo.

FcγRIV is expressed on the cell membrane of splenic and bone marrow dendritic cells, monocytes, and macrophages as well as peripheral blood monocytes, neutrophils, thioglycollate-elicited macrophages, and myeloid cells. FcγRIV is absent from lymphoid populations, T cells, B cells, NK cells, and other granulocytes.

Fcγ receptors are the primary mediators of IgG effector responses, and individual Fc receptors (FcR) have different affinities for different IgG subclasses¹. Four FcγRs are present in mice², and FcγRIV (FcγRL3, CD16.2) binds to IgG2a, IgG2b³, and IgE⁴, but not IgG1 or IgG3³. FcγRIV is a high-affinity receptor for monomeric IgG2a and IgG2b and a low-affinity IgE receptor for both IgE^a and IgE^b, binding to aggregates but not monomers⁴. Additionally, IgE immune complexes can displace IgG2 from FcγRIV. Surface expression of FcγRIV requires γ chain coexpression in vitro and in vivo³. FcγRIV and the γ chain are upregulated on bone marrow-derived monocytes by IFN-γ and LPS and are downregulated by TGF-β and IL-4.

According to surface plasmon resonance, 9E9 has strong reactivity to FcγRIV as well as low level binding to FcγRII and FcγRIII². In vivo, 9E9 binds and blocks FcγRIII only when 9E9 first binds FcγRIV on the same effector cell, resulting in concurrent inhibition of FcγRIII and FcγRIV. Native 9E9 binds to FcγRII and FcγRIII via the Fc.

9E9 was produced by immunizing Armenian hamsters with an FcγRIV-IgG1 fusion protein consisting of the extracellular domain of FcγRIV fused to a mouse IgG1 Fc portion (D265A-variant deficient in Fc-receptor binding)³. Splenic B cells were then fused to a mouse fusion partner, and hybridoma clones were screened for binding to CHO-K1-FcγRIV cells expressing FcγRIV.

Blocking studies with 9E9 show that FcγRIV is necessary for IgG2a and IgG2b mediated platelet clearance in vivo¹. Additionally, blocking FcγRIV with 9E9 reduces B-cell depletion². 9E9 also interferes with immune complex binding to FcγRIV³ and can block FcγRIII on macrophages and neutrophils².

1. Nimmerjahn F, Ravetch JV. Science. 310(5753):1510-1512. 2005.
2. Tipton TR, Mockridge CI, French RR, et al. Blood. 126(24):2643-2645. 2015.
3. Nimmerjahn F, Bruhns P, Horiuchi K, et al. Immunity. 23(1):41-51. 2005.
4. Mancardi DA, Iannascoli B, Hoos S, et al. J Clin Invest. 118(11):3738-3750. 2008.
5. Castro-Dopico T, Dennison TW, Ferdinand JR, et al. Immunity. 50(4):1099-1114.e10. 2019.