Anti-VEGFR-2 scFvs for cell isolation. Single-chain antibodies recognizingthe human vascular endothelial growth factor receptor-2 (VEGFR-2/flk-1) onthe surface of primary endothelial cells and preselected. CD34(+) cells from cord blood

Five specific single-chain antibodies recognizing the human vascular endoth elial growth factor receptor-2 (VEGFR-2/KDR) were selected from a V-gene ph age display library constructed from mice immunized with the extracellular domain of VEGFR-2 (Tg-like domain 1-7), hll five scFv antibodies (A3, A7, B 11, G3, and H1) bound to the purified native antigen in enzyme-linked immun osorbent assay and Dot Blot, and showed no crossreactivity to the human VEG F-receptor 1 (VEGFR-1). The selected antibodies recognize a conformation-de pendent epitope of the native receptor and do not recognize denatured antig en in Western blots, as well as linear overlapping peptides comprising the sequence of the human VEGFR-2. The five scFv antibodies bind to the surface of endothelial cells overexpressing human VEGFR-2 c-DNA (PAE/VEGFR-2 cells ) as detected by surface immunofluorescence using confocal microscopy. In a ddition scFv A7 specifically detected VEGFR-2 expresring endothelial cells in the glomerulus of frozen human kidney tissue sections. Therefore, A7 has potential clinical application as a marker for angiogenesis in cryosection s of different human tissues. Additionally, two recombinant scFvs (A2 and A 7) very efficiently recognize VEGFR-2 on PAE/VEGFR-2 cells and freshly prep ared human umbilical vein endothelial cells by fluorescence-activated cell sorter (FACS) analysis. The scFv fragment A7, which was the most sensitive antibody in FAGS analysis, recognizes human CD34*VEGFR-2* hematopoietic imm ature cells within the population of enriched CD34(+) cells isolated from h uman cord blood. The dissociation constant of A7 mas determined to be K-d = 3.8 x 10(-9) M by BIAcore analysis, In conclusion, scFv fragment A7 scents to be an important tool for FAGS analysis and cell sorting of vascular end othelial cells, progenitor cells and hematopoitic stem cells, which are pos itive for VEGFR-2 gene expression.