Anti-VEGF [G6-31] | Absoluteantibody貨號Ab01022-2-0
Fc Silent™ Antibodies: Turning down the background noise 只要訊號不要雜訊 (Engineered Fc Domain)
Species and Isotype: Mouse IgG2a, kappa
Clone Number: G6-31
UniProt Accession Number of Target Protein: Q00731
Published Application(s): ELISA; crystallisation; block; SPR; IF
Published Species Reactivity: Mouse
Immunogen: The anti-mVEGF Fab G6 underwent light chain randomisation by placing stop codons in positions 91-96 in CDR-L3 and an equimolar mix of oligonucleotides designed for mutagenesis was annealed to the mV401 stop template phagemid, followed by mutagenesis and E. colielectroporation. Binding selection was performed with mVEGF immobilised on immunoplates followed by solution-phase sorting with increasing stringency and single-point competetive ELISA to screen for high-affinity clones. The variable domain was then cloned into vectors for Fab expression inE. coli.
Specificity: G6-31 recognises a VEGF epitope which is conserved between human and mouse VEGF and overlaps with the receptor binding surface. The KD of G6-31 binding to m-VEGF and h-VEGF is ~0.9 and 1.5 nM respectively. Alanine-scanning mutagenesis of solvent-accessible residues of h-VEGF identified 10 residues that result in >4-fold loss in binding affinity including F17, M18, Y21, Q22, Y25, I83,H86, Q89 and I91.
Application Notes: A VEGF phage ELISA was used to measure binding affinity of G6-31 to individual alanine-substituted VEGF mutants versus wild type VEGF. Crystals of G6-31 and the receptor binding fragment of human VEGF have been solved at 3.6 Å (Fuh et al, 2005). Treatment of mice undergoing E2-induced PRLoma with G6-31 was used to block VEGF-A, causing reductions in the Ki-67-positive anterior cells, increases in TUNEL-positive anerior cells, and repair of the microvessel count by CD34-immunohistochemistry (Miyajima et al, 2010). Fluorescence quenching solution assay and SPR have been used to determine Kon and Koff at 25 degrees celsius (Lee et al, 2004).
Antibody First Published in:
Fuh et al Structure-function studies of two synthetic anti-vascular endothelial growth factor Fabs and comparison with the Avastin Fab. J Biol Chem. 2006 Mar 10;281(10):6625-31 PMID:16373345 Note on publication: Describes the isolation of the antibody from synthetic antibody phage libraries and analysis of VEGF binding.