MRSA-PBP2a单克隆抗体乳胶凝集检测法的建立  

Establishment of a Latex Agglutination Test Using Anti-PBP2a Monoclonal Antibody for Detection of MRSA-PBP2a

作　　者：徐霞[1] 陈思聪[1] 唐晓华[1] 

机构地区：[1]广州医学院检验系,广东广州510182

出　　处：《中山大学学报（医学科学版）》2008年第5期611-614,628,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广州市医药卫生科技项目(2005-YB-135);广州医学院项目(03-K-32)

摘　　要：【目的】应用抗PBP2a单克隆抗体,以化学交联法致敏羧化聚苯乙烯乳胶,建立快速检测MRSA-PBP2a的乳胶凝集法。【方法】以辛酸-硫酸铵法纯化PBP2a单克隆抗体,将纯化后单克隆抗体和羧化乳胶经碳化二亚胺(EDC)共价偶联,并对偶联条件(偶联抗体浓度、乳胶浓度、偶联时间和偶联缓冲液)进行探索和优化,建立检测PBP2a的乳胶凝集法。【结果】140mg/L的抗体浓度与15g/L的羧化聚苯乙烯乳胶,在pH 5.8缓冲液中偶联8h后有较好的凝集效果和偶联效率,所建立的乳胶凝集法敏感性和特异性良好。【结论】初步建立了检测PBP2a的胶乳凝集法,为研制MRSA快速鉴定试剂盒奠定了良好的基础。[Objective] To develop a simple and rapid latex agglutination test （LAT） for the detection of MRSA-PBP2a, which is based on Carboxyl-polystyrene latex sensitized with anti-PBP2a monoclonal antibody （mAb） by chemical cross-linking method. [ Methods ] The purification of mAb from ascites fluids was performed by caprylic acid/ammonium sulfate precipitation （CA-AS）, the purified mAb was covalently coupled to carboxylate latex by the carbodiimide method, then the coupling conditions such as coupling antibody concentration, the coupling time, the latex concentration and the coupling buffer were studied to optimize the latex agglutination test （LAT） for the detection of MRSA-PBP2a. [Results] The best agglutination and the highest coupling efficiency was reached when the antibody concentration was 140 mg/L and the latex concentration was 1.5 g/L with the reaction time of 8 hours and the buffer of pH=5.8. The sensitivity and specifity of the LAT in detecting the PBP2a were high. [Conclusion] A LAT for detection of PBP2a was successfully established, which lays the foundation for the preparation of a LAT kit.

关 键 词：乳胶凝集法 单克隆抗体 检测法 PBP2A 辛酸-硫酸铵法 共价偶联 抗体浓度 聚苯乙烯 

分 类 号：R446.11[医药卫生—诊断学] R392-33[医药卫生—临床医学]