United Kingdom (UK)
Recombinant monoclonal antibody to Kit . Manufactured using AbAb’s Recombinant Platform with variable regions (i.e. specificity) from the phage display antibody 79D.
UniProt Accession Number of Target Protein: P10721
Alternative Name(s) of Target: Mast/stem cell growth factor receptor Kit (EC:2.7.10.1); SCFR; Piebald trait protein; PBT; Proto-oncogene c-Kit; Tyrosine-protein kinase Kit; p145 c-kit; v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog; CD117
Immunogen: Phage pools consisting of a phage-displayed synthetic antibody library (library F) were cycled through five rounds of selections by using KITD4-5.
Specificity: The antibody is specific for KIT, and it binds to the membrane-proximal Ig-like D4 of KIT. The receptor tyrosine kinase KIT is a transmembrane protein that plays crucial roles in mediating diverse cellular processes, including cell differentiation, proliferation, and cell survival, among other activities.
Application Notes: The crystal structure of the Fab in complex with KIT D4D5 fragment was determined. Surface plasmon resonance analysis was employed to quantitatively characterize the kinetics and dissociation constants of the Fab (Kd = 6.4 pM). 3T3 cells expressing WT KIT receptor were incubated with the IgG and Fab versions of the antibody following stimulation with SCF. Lysates of unstimulated or SCF-stimulated cells were subjected to immunoprecipitation with the IgG and Fab followed by SDS/PAGE and immunoblotting with IgG and Fab. SCF-stimulated autophosphorylation of KIT was strongly inhibited upon binding of both fragments (5 nM for Fab and 1 nM for IgG). The IgG and Fab fragments inhibited the proliferation of KIT-Dependent Ba/F3 cells. The Fab fragment showed inhibition of cell growth at 5 nM, while the IgG fragment at 10 nM (Reshetnyak et al. 2013; PMID: 24127596).
Antibody first published in:
Reshetnyak et al. Structural basis for KIT receptor tyrosine kinase inhibition by antibodies targeting the D4 membrane-proximal region. Proc Natl Acad Sci U S A. 2013 Oct 29; 110(44): 17832–17837. PMID:24127596
Note on publication:
The paper describes the generation and characterization of a panel of inhibitory KIT antibodies.
Flow cytometry using anti-Kit antibody 79D (Ab03168). HEK293 cells were stained with the anti-unknown specificity antibody (Ab00178-23.0; isotype control, black line) or the rabbit IgG version of 79D (Ab03168-23.0, blue line) at a dilution of 1:100 for 1h at RT. After washing, the bound antibody was detected using a goat anti-rabbit IgG AlexaFluor® 488 antibody at a dilution of 1:1000, and the cells were analyzed using a FACSCanto flow cytometer.
Immunofluorescence staining of HEK293 cells with anti-Kit antibody 79D (Ab03168). Immunofluorescence analysis of paraformaldehyde fixed HEK293 cells stained with the chimeric rabbit IgG version of 79D (Ab03168-23.0) (1:100 dilution) for 1h followed by Alexa Fluor® 488 secondary antibody (1:1000 dilution), showing membrane staining. The nuclear stain is DAPI (blue). Panels show, from left-right, top-bottom, Ab03168-23.0, DAPI, merged channels, and an isotype control. The isotype control was an unknown specificity antibody (Ab00178-23.0) followed by staining with Alexa Fluor® 488 secondary antibody.