人C-反应蛋白磁微粒化学发光酶免疫测定法的建立  

Development of Magnetic Particle-based Chemiluminescence Enzyme Immunoassay for Human C-reactive Protein

作　　者：罗梦洁 肖铎 曾璇 谭楚帆 徐叶 钟志宏[1] 刘如石[1,3,4] 郑姣 LUO Mengjie;XIAO Duo;ZENG Xuan;TAN Chufan;XU Ye;ZHONG Zhihong;LIU Rushi;ZHENG Jiao(School of Medicine,Hunan Normal University,Changsha 410013,Hunan,China;GI Medicine of Xiangtan County People’s Hospital,Xiangtan 411228,Hunan,China;Hunan Xuxiang Biotechnology Co.,Ltd.,Changsha 410221,Hunan,China;Immunodiagnostic Reagents Engineering Research Center of Hunan Province,Changsha 410013,Hunan,China)

机构地区：[1]湖南师范大学医学院,中国湖南长沙410013 [2]湘潭县人民医院消化内科,中国湖南湘潭411228 [3]湖南旭翔生物科技有限公司,中国湖南长沙410221 [4]免疫诊断试剂湖南省工程研究中心,中国湖南长沙410013

出　　处：《生命科学研究》2024年第2期135-142,151,共9页Life Science Research

基　　金：国家自然科学基金青年基金项目(81702005);湖南省自然科学基金项目(2019JJ50370)。

摘　　要：人C-反应蛋白(C-reactive protein,CRP)是炎症以及各种相关疾病如病毒感染、心血管疾病等诊断、治疗和预后的临床检测指标。为了建立一种快速、准确的CRP定量免疫测定方法,将表达纯化的重组CRP作为抗原免疫小鼠,获得了5株稳定分泌抗体的单克隆抗体细胞株,采用双抗体夹心酶联免疫吸附测定法(double antibody sandwich enzyme-linked immunosorbent assay,DAS-ELISA)初步鉴定筛选的抗人CRP单克隆抗体,并分别选择mAb 9D6和mAb 9G4作为捕获抗体与检测抗体,建立用于人CRP检测的化学发光酶免疫测定法(chemiluminescence enzyme immunoassay,CLEIA),最后通过测定分析临床血清CRP样本,评价CLEIA的性能。结果显示,基于9D6/9G4-AP单克隆抗体对的CLEIA测定范围为0.1767~500μg/L(可扩展至100 mg/L);所建立的CLEIA与医院采用的免疫散射比浊法(R^(2):0.9496,P<0.0001)表现出良好的相关性,且Bland-Altman分析中96.36%(106/110)的点在95%一致性界限范围内显示两种检测方法具有较好的一致性。结果初步表明,建立的分析方法在临床诊断中具有较好的应用前景。Human C-reactive protein(CRP)is an excellent clinical biomarker for the diagnosis,treatment and prognosis of inflammation and various related diseases such as viral infections and cardiovascular diseases.Herein,the expressed recombinant CRP as an antigen was used to immunize mice for establishing a rapid and accurate quantitative immunoassay for CRP.Five hybridoma cell lines for stable monoclonal antibody production were obtained.The developed monoclonal antibodies(mAbs)screened against human CRP were initially identified by a double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA),and then mAb 9D6 and mAb 9G4 were selected as the capture and detection antibodies,respectively,to estab-lish the magnetic particle-based chemiluminescence enzyme immunoassay(CLEIA)for detection of CRP.Fi-nally,the analytical performance of the established CLEIA was evaluated by measuring CRP in clinical serum samples.The results showed that CLEIA based on the monoclonal antibody pair of 9D6/9G4-AP was charac-terized by the measurement interval from 0.1767μg/L to 500μg/L(extended to 100 mg/L),and had an excel-lent correlation with the immune scattering turbidimetry commonly used in hospitals(R^(2):0.9496,P<0.0001).In the Bland-Altman analysis,96.36%(106/110)of the points were within the consistency limit of 95%,in-dicating that the two detection methods had good consistency.These results preliminarily showed that the developed analytical method may have a good application prospect in clinical diagnosis.

关 键 词：C-反应蛋白(CRP) 单克隆抗体(mAb) 双抗体夹心酶联免疫吸附测定法(DAS-ELISA) 化学发光酶免疫测定法(CLEIA) 

分 类 号：Q503[生物学—生物化学] R446.6[医药卫生—诊断学]