D2蛋白酶间接ELISA检测方法的建立  

Development of An Indirect ELISA Method for D2 Protease

作　　者：苏洁[1] 王斌[1] 鲁晓晴[1] 赵巍[1] 闫志勇[1] 钱冬萌[1] 丁守怡[1] 宋旭霞[1] 杨丽[1] 刘海燕[1] 

机构地区：[1]青岛大学医学院微生物学教研室青岛市医药生物技术重点实验室,山东青岛266071

出　　处：《中国生物制品学杂志》2010年第2期207-210,共4页Chinese Journal of Biologicals

基　　金：国家‘973’计划重大基础研究前期研究专项(2004-CCA02400);泰山学者工程资助

摘　　要：目的建立D2蛋白酶间接ELISA检测方法,为D2蛋白酶的进一步研究及应用奠定基础。方法以纯化的D2蛋白酶为抗原,免疫新西兰白兔,制备D2蛋白酶多克隆抗体,建立D2蛋白酶间接ELISA检测方法,并进行验证及初步应用。结果D2蛋白酶间接ELISA检测方法的最佳反应条件为:一抗稀释度为1∶64000,二抗稀释度为1∶80000,抗原稀释度为1∶100,饱和包被浓度为1160ng/ml,封闭剂为10%胎牛血清。标准曲线的线性检测范围为18～1160ng/ml。该方法准确性、重复性良好,特异性强、敏感性高,与琼脂扩散试验检测结果的符合率为95.5%。结论已建立了D2蛋白酶间接ELISA检测方法,可用于D2蛋白酶含量的检测。Objective To develop an indirect ELISA method for D2 protease and lay a foundation of further study and application of the protease.Method Purified D2 protease was used as an antigen for immunization of New Zealand white rabbits to prepare the polyclonal antibody against D2 protease,based on which an indirect ELISA method for D2 protease was developed,verified and preliminarily applied.Results The optimal dilutions of the first and the second antibodies used for the developed ELISA were 1∶64 000 and 1∶80 000 respectively,while that of antigen was 1∶100.The saturated antigen concentration for coating was 1 160 ng/ml, and 10%fetal calf serum was used as blocking agent.The linear range of standard curve of the developed ELISA was 181 160 ng/ml. The developed ELISA showed high accuracy,reproducibility,specificity and sensitivity.The coincidence rate of detection results by the developed ELISA and by agar diffusion test was 95.5%.Conclusion An indirect ELISA method suitable for determination of D2 protease content was developed.

关 键 词：D2蛋白酶 间接ELISA 多克隆抗体 

分 类 号：Q55[生物学—生物化学] R392.33[医药卫生—免疫学]