抗人Flt-1单克隆抗体的制备和活性鉴定

目的:制备小鼠抗人血管内皮生长因子受体-1(Flt-1)的单克隆抗体(mAb),并鉴定其免疫学特性。方法:以重组人Flt-1胞外Ⅲ区蛋白为抗原,通过经典的杂交瘤制备和活性筛选方法建立能稳定分泌抗Flt-1蛋白的mAb杂交瘤细胞株。结果:经传统的小鼠腹水型抗体的制备和纯化方法获得了高纯度的抗人Flt-1胞外Ⅲ区蛋白的mAb。ELISA方法测定出该抗体的免疫球蛋白亚型为IgG1,轻链为κ链。West-ern blot结果显示该抗体能特异性地识别重组人Flt-1胞外Ⅲ区蛋白,FACS结果表明该抗体能结合到Flt-1阳性的脐静脉内皮细胞和K562/A02细胞表面,并呈现出抗体浓度依赖性。结论:成功建立了1株能够稳定分泌抗重组人Flt-1胞外Ⅲ区蛋白的mAb细胞株XA12,为今后进一步开展Flt-1及其胞外Ⅲ区蛋白的生物学功能研究以及基因工程抗体研究奠定了基础。

Preparation and identification of monoclonal antibody against human Flt-1

AIM: To prepare functional monoclonal antibodies(mAb)against recombinant human Flt-1(rhFlt-1).METHODS: A cell line stable secreting mAb was established by using FLT-1 extracellular domain III as antigen and hybridoma technique.Then it was purified in large-scale from mouse ascites by protein G affinity chromatography.The characteristics of mAb were then determined by ELISA,Western blotting and FACS.RESULTS: The immunoglobin subtype of mAb XA12 was IgG1 with kappa(κ) light chains,and it could recognize rhFlt-1 specifically.Furthermore,mAb XA12 could bind to rhFlt-1with high affinity(K=1.28±0.05 nmol/L).It could also be used to detect Flt-1-positive cells,such as human umbilical vascular endothelial cells(HUVECs) and K562/A02 in a dose-dependent fashion.CONCLUSION: A hybridoma cell line secreting functional anti-rhFlt-1 mAb was successfully prepared.The antibody can be used to study the function of Flt-1 and further potentially optimized for clinical purpose.