抗人Flt-1单克隆抗体的制备和活性鉴定

Preparation and identification of monoclonal antibody against human Flt-1

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。

AIM： To prepare functional monoclonal anti- bodies （mAb） against recombinant human Fit-1 （ rhFIt-1 ）. METHODS： A cell line stable secreting mAb was estab- lished 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 （g） light chains, and it could recognize rhFIt-1 specifically. Further- more, mAb XA12 could bind to rhFIt-lwith high affinity （K= 1.28 ＋0.05 nmol/L）. It could also be used to detect Fit-I- 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-rhFIt-1 mAb was successfully prepared. The antibody can be used to study the function of Fit-1 and further potentially optimized for clinical purpose.