Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor Ⅱ

AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domain Ⅲ of vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Soluble KDR Ig domain III (KDR-Ⅱ) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-Ⅲ were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial cells. RESULTS: A monoclonal antibody, YcomlD3 (IgGl), was generated from a mouse immunized with the recombi-nant KDR-Ⅲ protein. YcomlD3 bound specifically to both the soluble KDR-Ⅲ and the cell-surface expressed KDR. YcomlD3 effectively bloc