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人C-反应蛋白磁微粒化学发光酶免疫测定法的建立

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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0.176 7~500 μg/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(R2:0.949 6,P<0.000 1)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.
Development of Magnetic Particle-based Chemiluminescence Enzyme Immunoassay for Human C-reactive Protein
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 0.176 7 μg/L to 500 μg/L(extended to 100 mg/L),and had an excel-lent correlation with the immune scattering turbidimetry commonly used in hospitals(R2:0.949 6,P<0.000 1).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.

C-reactive protein(CRP)monoclonal antibody(mAb)double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA)chemiluminescence enzyme immunoassay(CLEIA)

罗梦洁、肖铎、曾璇、谭楚帆、徐叶、钟志宏、刘如石、郑姣

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湖南师范大学医学院,中国湖南 长沙 410013

湘潭县人民医院消化内科,中国湖南 湘潭 411228

湖南旭翔生物科技有限公司,中国湖南 长沙 410221

免疫诊断试剂湖南省工程研究中心,中国湖南 长沙 410013

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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)

国家自然科学基金青年基金湖南省自然科学基金

817020052019JJ50370

2024

生命科学研究
湖南师范大学

生命科学研究

CSTPCD
影响因子:0.421
ISSN:1007-7847
年,卷(期):2024.28(2)
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