心传导束相关特异性抗体制备及其在心脏疾病检测中的初步应用  

Preparation of specific antibody associated with cardiac conduction bundles and its preliminary application in heart disease detection

作　　者：任帆 陆辉 李耀 张昕霞 潘红阳 毛峥嵘[1,3] 周韧 Ren Fan;Lu Hui;Li Yao;Zhang Xinxia;Pan Hongyang;Mao Zhengrong;Zhou Ren(Department of Pathology and Pathophysiology,School of Medicine,Zhejiang University,Institute of Pathology and Forensic Medicine,Zhejiang University,Hangzhou 310058;Yikang(Hangzhou)Biotechnology Co.,LTD,Hangzhou 310015;Center for Forensic Expertise,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学医学院病理学与病理生理学系,病理学与法医研究所,杭州310058 [2]宜康(杭州)生物技术有限公司,杭州310015 [3]浙江大学司法鉴定中心,杭州310058

出　　处：《解剖学杂志》2022年第1期11-16,F0003,共7页Chinese Journal of Anatomy

摘　　要：目的:研发和制备心钠肽(ANP)的兔单克隆抗体,检测其在心窦房结、传导束的免疫特性表达及其在心脏疾病中的检测应用。方法:采用固相多肽合成法合成ANP特异性多肽,对新西兰大耳白兔进行免疫,取出兔脾细胞与兔的骨髓瘤细胞240E-W3进行融合,采用兔单克隆抗体技术制备兔单抗,运用免疫印迹、免疫组织化学法鉴定其特异性和敏感性。收集24例人心组织,其中呼吸系统疾病、心血管系统疾病、其他疾病导致死亡的比例分别占33%、54%、13%,对24例人心组织进行免疫组织化学检测ANP表达。结果:制备抗ANP的兔单克隆抗体(ab225873),经免疫印迹检测显示,ANP在人心组织中可以特异性识别大小为16 kD的条带。免疫组织化学结果显示,ANP特异性表达在成人心组织的窦房结和左心室的传导束,但心室肌以及成人的其他组织中不表达。24例人心组织中心房肌阳性率为100%,窦房结阳性率为100%,传导束阳性率为50%,心室肌阳性率为0。结论:本研究制备的抗ANP兔单克隆抗体(ab225873)在免疫印迹和免疫组织化学上均表现有良好的特异性与敏感性,能特异识别心窦房结、传导束,为研究窦房结、传导束相关疾病提供分子检测工具。Objective:To develop and prepare a rabbit monoclonal antibody against natriuretic peptides A(ANP),to identify its immune characteristics in the sinoatrial node and conduction bundles of the heart,and to explore its expression and application in cardiac diseases.Methods:New Zealand white rabbits were immunized with ANP-specific peptides synthesized by solid-phase peptide method.Then rabbit spleen cells were taken out and fused with rabbit myeloma cells 240 E-W3.Rabbit monoclonal antibody technology was used to develop rabbit monoclonal antibodies.The specificity and sensitivity of ANP antibody was identified by using Western blotting and immunohistochemistry.Cardiac tissues were collected from 24 people,of which 33%died due to respiratory diseases,54%died due to cardiovascular diseases,and 13%died due to other diseases.Immunohistochemistry was also performed on these cardiac tissues from 24 human cases.Results:An anti-ANP rabbit monoclonal antibody(ab225873)was developed.The Western blotting results showed that anti-ANP specifically recognized a band with a size of 16 kD in human heart tissue.Immunohistochemistry results showed that anti-ANP was specifically expressed in the sinoatrial node of human adult heart tissues and conduction bundles of left ventricle,but not in the ventricular muscle or other human tissues.The 24 human heart tissues were 100%positive for atrial muscle,100%positive for sinoatrial node,50%positive for conduction bundle,and 0 positive for ventricular muscle.Conclusion:The anti-ANP rabbit monoclonal antibody(ab225873)developed in this study has good specificity and sensitivity on both Western blotting and immunohistochemistry.It specifically recognizes the sinoatrial node and conduction bundles of heart tissues,which provides a molecular detection tool for the study of sinoatrial node and conduction bundle-related diseases.

关 键 词：心钠肽 窦房结 心传导束 兔单克隆抗体 免疫印迹 免疫组织化学 

分 类 号：R541[医药卫生—心血管疾病]