血清C反应蛋白荧光免疫层析检测方法的建立  

Quantitation Determination of CRP in Serum with Fluorescence Immune Chromatography

作　　者：葛宇新 丁秋雨 钟晓骝 栗慧超 刘倩[3] 史新宇[3] 赵晶晶[3] 于源华[3] 张昊[3] GE Yuxin DING Qiuyu ZHONG Xiaoliu LI Huichao LIU Qian SHI Xinyu ZHAO Jingjing YU Yuanhua ZHANG Hao(Hospital of Changchun University of Science and Technology, Changchun 130022 School of Chemistry and Environmental Engineering, Changchun University of Science and Technology, Changchun 130022 School of Life Science and Technology, Changehun University of Science and Technology, Changchun 130022)

机构地区：[1]长春理工大学校医院,长春130022 [2]长春理工大学化学与环境工程学院,长春130022 [3]长春理工大学生命科学技术学院,长春130022

出　　处：《长春理工大学学报（自然科学版）》2016年第6期129-133,共5页Journal of Changchun University of Science and Technology(Natural Science Edition)

基　　金：吉林省科技发展计划项目(20140309013YY)

摘　　要：针对人体急性炎症反应的快速检测,建立了基于量子点碲化镉（Cd Te）的荧光免疫层析分析方法及快速定量检测卡,实现血清中炎症标志物C反应蛋白（CRP）的快速定量检测。该检测卡采用双抗体夹心法,首先利用酶联免疫方法筛选出CRP测定最佳包被抗体与标记抗体,然后通过N-羟基琥珀酰（NHS）和1-乙基-3-（3-二甲氨基丙基）碳二亚胺盐酸盐（EDC）共价偶联法,将Cd Te量子点与CRP鼠源单克隆抗体偶联,制备抗体荧光标记物,同时优化不同条件下单克隆抗体与Cd Te量子点的偶联效果,琼脂糖凝胶电泳进行鉴定,进而构建CRP荧光免疫层析检测卡,通过建立量子点荧光强度与CRP标准品浓度之间的定量关系,从而实现人体血清中CRP的定量检测。结果表明,CRP检测卡定量检测线性范围为0.1~1000ng/ml,临床样本测试结果显示与进口试剂具有良好的相关性。因此研究为急性炎症反应的快速诊断,及荧光免疫层析检测卡的研发提供了技术基础。For rapid detection of human acute inflammatory response,a fluorescence immuno-chromatographic analysis method of double monoclonal antibody sandwich ELISA was developed,which based on quantum dots of cadmium telluride（Cd Te）,for the rapid and quantitative detection of inflammatory marker C-reactive protein（CRP） in serum. In this work,at first,capture antibody and labeled antibody for CPR detection were screened by ELISA. Second,Cd Te quantum dots and CRP murine monoclonal antibodies were covalent binding by N-hydroxysuccinimide（NHS） and1-ethyl-3-（3-dimethylaminopropyl） carbodiimide hydrochloride（EDC）,as well as the best condition of covalent binding was optimized and identified by agarose gel electrophoresis. After that,fluorescent immuno-chromatographic test strip for CPR rapid detection was prepared. In addition,the quantitative relationship between the quantum dot fluorescence intensity and the concentration of CRP standard was established,the results showed that the linear range of quantitative determination of CRP test strip was from 0.1 to 1000ng/ml,the experimental test strip and commercial reagents were used in clinical serum samples detection,the results showed a good correlationship. In a word,this work can provide valubale basis for the rapid diagnosis of acute inflammatory reactions and the development of fluorescence immunoassay chromatography test strip.

关 键 词：C反应蛋白 量子点 荧光免疫层析 定量检测 

分 类 号：R375[医药卫生—病原生物学]