一株鼠抗人CD36单克隆抗体的制备及应用

doi:10.3969/j.issn.1000-8535.2020.04.006

广州医药 ›› 2020, Vol. 51 ›› Issue (4): 28-31.DOI: 10.3969/j.issn.1000-8535.2020.04.006

一株鼠抗人CD36单克隆抗体的制备及应用

陈大伟, 徐秀章, 夏文杰, 邵媛, 王嘉励, 刘静, 邓晶, 陈扬凯, 丁浩强, 叶欣

广州血液中心(广州 510095)

收稿日期:2020-03-12 出版日期:2020-07-20 发布日期:2021-11-28
通讯作者: 叶欣,E-mail:huang_yexin@qq.com
基金资助:
广州市医药卫生科技项目(20171A011281); 国家青年自然科学基金(81601451);广东省自然科学基金(2016A030313124)

Preparation and application of a mouse monoclonal antibody against human CD36

CHEN Dawei, XU Xiuzhang, XIA Wenjie, SHAO Yuan, WANG Jiali, LIU Jing, DENG Jing, CHEN Yangkai, DING Haoqiang, YE Xin

Guangzhou Blood Center,Guangzhou 510095,China

Received:2020-03-12 Online:2020-07-20 Published:2021-11-28

摘要/Abstract

摘要： 目的本研究通过已建立的稳定表达人血小板CD36的HEK293T细胞系,制备鼠源的抗人CD36单克隆抗体并进行特性鉴定。方法利用已经建立的稳定表达人血小板CD36的HEK293T细胞系对CD36(-/-)C57小鼠进行免疫,取脾脏与小鼠骨髓瘤细胞融合,筛选出阳性克隆。纯化单克隆抗体后,利用Western blot检测抗体结合活性。利用小鼠IgG类/单抗亚型鉴定试剂鉴定单克隆抗体的抗体亚型。通过流式细胞检测和血小板抗原单克隆抗体特异性免疫固定检测(MAIPA)鉴定其诊断应用价值。结果经筛选后得到一株杂交瘤细胞,抗体亚型为IgG2a,轻链为κ链,Western blot试验表明该单克隆抗体特异性识别人血小板CD36抗原。MAIPA结果显示,与商业化单克隆抗体FA6-152相比,该单克隆抗体灵敏度更高。结论成功制备了一种鼠抗人CD36单克隆抗体,为临床鉴定CD36抗体,筛选CD36阴性献血员提供了新的工具,也为今后进一步研究CD36在血液免疫疾病中作用机制提供了实验基础。

关键词: CD36, 单克隆抗体, 制备, 鉴定, MAIPA

Abstract: Objective In this study, by the established HEK293T cell line with stable expression of human platelet CD36, murine anti-human CD36 monoclonal antibody was prepared and characterized. Methods The established HEK293T cell line with stable expression of human platelet CD36 was used to immunize CD36 (-/-) C57 mice, and the spleen was fused with mouse myeloma cells to screen for positive cloning.After the purification of monoclonal antibody, the antibody binding activity was detected by Western blot.Mouse rapid antibody isotyping reagent was used to identify the subtype of monoclonal antibody.Its diagnostic value was evaluated by flow cytometry and monoclonal antibody-specific immobilization of platelet antigen (MAIPA). Results After screening, a high-producing hybridoma cell was obtained, the subtype of monoclonal antibody was IgG2a and the light chain was κ chain. Western blot analysis showed that the monoclonal antibody could specifically recognize CD36 antigen of human platelet. MAIPA results showed that the monoclonal antibody was more sensitive than the commercial monoclonal antibody FA6-152. Conclusion It is concluded that a mouse anti-human CD36 monoclonal antibody with biological activity has been obtained, which provides a new tool for the clinical identification of CD36 antibody and the screening of CD36 negative blood donors, and also provides an experimental basis for further research on the mechanism of CD36 in blood immune diseases.

Key words: CD36, Monoclonal antibody, Preparation, Identification, MAIPA

陈大伟, 徐秀章, 夏文杰, 邵媛, 王嘉励, 刘静, 邓晶, 陈扬凯, 丁浩强, 叶欣. 一株鼠抗人CD36单克隆抗体的制备及应用[J]. 广州医药, 2020, 51(4): 28-31.

CHEN Dawei, XU Xiuzhang, XIA Wenjie, SHAO Yuan, WANG Jiali, LIU Jing, DENG Jing, CHEN Yangkai, DING Haoqiang, YE Xin. Preparation and application of a mouse monoclonal antibody against human CD36[J]. Guangzhou Medical Journal, 2020, 51(4): 28-31.

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一株鼠抗人CD36单克隆抗体的制备及应用

Preparation and application of a mouse monoclonal antibody against human CD36

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