抗人Flt-1单克隆抗体的制备与鉴定
[Preparation and identification of monoclonal antibody against human Flt-1].
作者信息
Xie Yin-liang, Ma Li, Qin Li, Liu Peng, Cao Shan-nan, Zhu Hai-yan, Xiong Dong-sheng, Xu Yuan-fu
机构信息
Chinese Academy of Medical Sciences, Tianjin, China.
出版信息
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 May;28(5):524-8.
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.
目的
制备抗重组人Flt-1（rhFlt-1）的功能性单克隆抗体（mAb）。
方法
以FLT-1细胞外结构域III为抗原，采用杂交瘤技术建立稳定分泌单克隆抗体的细胞系。然后通过蛋白G亲和层析从鼠腹水中大规模纯化该抗体。接着采用酶联免疫吸附测定（ELISA）、蛋白质印迹法（Western blotting）和荧光激活细胞分选术（FACS）对单克隆抗体的特性进行测定。
结果
单克隆抗体XA12的免疫球蛋白亚型为IgG1，轻链为κ链，能特异性识别rhFlt-1。此外，单克隆抗体XA12能以高亲和力（K = 1.28±0.05 nmol/L）与rhFlt-1结合。它还能以剂量依赖的方式用于检测Flt-1阳性细胞，如人脐静脉内皮细胞（HUVECs）和K562/A02。
结论
成功制备了分泌功能性抗rhFlt-1单克隆抗体的杂交瘤细胞系。该抗体可用于研究Flt-1的功能，并可能进一步针对临床目的进行优化。
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