抗人血管内皮钙黏蛋白5单克隆抗体的制备与应用
[Preparation and application of monoclonal antibody against human vascular endothelial cadherin 5].
作者信息
Ding Jie, He Yang, Yang Jianfeng, Zuo Bin, Yang Binghua
机构信息
Clinical Laboratory, First Affiliated Hospital, Soochow University, Suzhou 215006, China.
Clinical Laboratory, First Affiliated Hospital, Soochow University; Jiangsu Provincial Institute of Hematology, Suzhou 215006, China.
出版信息
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2016 Jan;32(1):106-11.
OBJECTIVE
To prepare and identify monoclonal antibody (mAb) against human vascular endothelial cadherin 5 (VECDH5).
METHODS
BALB/c mice were immunized with recombinant VECDH5 protein for preparing mAb using hybridoma technique. The positive clones were confirmed and selected by indirect ELISA for titer determination. Western blotting, flow cytometry, immunofluorescence staining, immunohistochemical staining were performed to identify the specificity and epitope.
RESULTS
One hybridoma cell strain secreting specific mAb against VECDH5 was obtained (2C11). ELISA showed that the titer of the ascites was 1:10 000. Western blotting, flow cytometry, immunofluorescence, immunohistochemistry demonstrated that the mAb could specifically recognize and bind VECDH5. Epitope identification showed that the amino acid sequence recognized by 2C11 was LDREVVPWYNLTVEA.
CONCLUSION
We have prepared mAb against human VECDH5, which has good binding ability and specificity.
目的
制备并鉴定抗人血管内皮钙黏蛋白5（VECDH5）的单克隆抗体（mAb）。
方法
用重组VECDH5蛋白免疫BALB/c小鼠，采用杂交瘤技术制备单克隆抗体。通过间接ELISA法确认并筛选阳性克隆以测定效价。进行蛋白质免疫印迹法、流式细胞术、免疫荧光染色、免疫组织化学染色以鉴定其特异性和表位。
结果
获得一株分泌抗VECDH5特异性单克隆抗体的杂交瘤细胞株（2C11）。ELISA显示腹水效价为1:10 000。蛋白质免疫印迹法、流式细胞术、免疫荧光、免疫组织化学表明该单克隆抗体可特异性识别并结合VECDH5。表位鉴定显示2C11识别的氨基酸序列为LDREVVPWYNLTVEA。
结论
我们已制备出抗人VECDH5的单克隆抗体，其具有良好的结合能力和特异性。
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