Böldicke T, Tesar M, Griesel C, Rohde M, Gröne H J, Waltenberger J, Kollet O, Lapidot T, Yayon A, Weich H
German Research Centre for Biotechnology, Department of Applied Genetics, Braunschweig, Germany.
Stem Cells. 2001;19(1):24-36. doi: 10.1634/stemcells.19-1-24.
Five specific single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) were selected from a V-gene phage display library constructed from mice immunized with the extracellular domain of VEGFR-2 (Ig-like domain 1-7). All five scFv antibodies (A2, A7, B11, G3, and H1) bound to the purified native antigen in enzyme-linked immunosorbent assay and Dot Blot, and showed no crossreactivity to the human VEGF-receptor 1 (VEGFR-1). The selected antibodies recognize a conformation-dependent epitope of the native receptor and do not recognize denatured antigen 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 addition scFv A7 specifically detected VEGFR-2 expressing endothelial cells in the glomerulus of frozen human kidney tissue sections. Therefore, A7 has potential clinical application as a marker for angiogenesis in cryosections of different human tissues. Additionally, two recombinant scFvs (A2 and A7) very efficiently recognize VEGFR-2 on PAE/VEGFR-2 cells and freshly prepared human umbilical vein endothelial cells by fluorescence-activated cell sorter (FACS) analysis. The scFv fragment A7, which was the most sensitive antibody in FACS analysis, recognizes human CD34+VEGFR-2+ hematopoietic immature cells within the population of enriched CD34+ cells isolated from human cord blood. The dissociation constant of A7 was determined to be K(d) = 3.8 x 10(-9) M by BIAcore analysis. In conclusion, scFv fragment A7 seems to be an important tool for FACS analysis and cell sorting of vascular endothelial cells, progenitor cells and hematopoitic stem cells, which are positive for VEGFR-2 gene expression.
从用血管内皮生长因子受体-2（VEGFR-2/KDR）胞外域（免疫球蛋白样结构域1-7）免疫的小鼠构建的V基因噬菌体展示文库中，筛选出5种特异性识别人类血管内皮生长因子受体-2的单链抗体。在酶联免疫吸附测定和斑点印迹中，所有5种单链抗体（A2、A7、B11、G3和H1）均与纯化的天然抗原结合，且与人血管内皮生长因子受体1（VEGFR-1）无交叉反应。所筛选的抗体识别天然受体的构象依赖性表位，在蛋白质印迹中不识别变性抗原，也不识别包含人类VEGFR-2序列的线性重叠肽段。通过共聚焦显微镜表面免疫荧光检测发现，这5种单链抗体与过表达人类VEGFR-2 cDNA的内皮细胞表面（PAE/VEGFR-2细胞）结合。此外，单链抗体A7能特异性检测冷冻保存的人肾组织切片肾小球中表达VEGFR-2的内皮细胞。因此，A7作为不同人类组织冰冻切片中血管生成的标志物具有潜在的临床应用价值。此外，通过荧光激活细胞分选仪（FACS）分析，两种重组单链抗体（A2和A7）能非常有效地识别PAE/VEGFR-2细胞和新鲜制备的人脐静脉内皮细胞上的VEGFR-2。单链抗体片段A7是FACS分析中最敏感的抗体，能识别从人脐带血分离的富集CD34+细胞群体中的人类CD34+VEGFR-2+造血未成熟细胞。通过生物传感器分析测定A7的解离常数为K(d)=3.8×10(-9)M。总之，单链抗体片段A7似乎是用于FACS分析以及对VEGFR-2基因表达呈阳性的血管内皮细胞、祖细胞和造血干细胞进行细胞分选的重要工具。
