斑点酶免疫渗滤法定量检测雌三醇的实验研究  

Establishment of the dot immunoenzyme filtration assay for quantitive detection of estriol

作　　者：薛添[1] 赵轶君[1] 董洁[1] 李红民[1] 

机构地区：[1]西北大学生命学院,陕西西安710069

出　　处：《细胞与分子免疫学杂志》2013年第8期874-876,881,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：陕西省教育厅重点实验室重点科研计划项目(11JS085);陕西省科技厅社会发展攻关项目(2011K12-61)

摘　　要：目的 制备高特异性的抗雌三醇（E3）单克隆抗体（mAb）, 建立操作简便、敏感、快速适用于基层使用的E3检测方法。方法 采用活化酯法制备偶联物E3-HRP、E3-BSA、3-OVA。应用mAb技术制备抗E3的mAb; 以硝酸纤维素膜为固相载体, 抗E3 mAb为包被抗体, 建立竞争抑制模式的斑点酶免疫渗滤法。结果 筛选出5株可分泌特异性抗E3 mAb的杂交瘤细胞株。mAb效价为1×10^5～5×10^5, 亲和常数为5.1×10^8～5.2×10^9 L/mol。检测敏感度为2 ng/mL, 检测范围2～200 ng/mL。结论 建立了检测速度快、灵敏度高、应用方便的斑点酶免疫渗滤法。Objective To make anti-estriol （E3） monoclonal antibody （mAb） with high specificity, and develop a rapid, simple and sensitive method for the detection of E3 in county-level institutions. Methods The compounds of E3 were conju-gated to horseradish peroxidase （HRP）, bonine serum albumin （BSA） and ovalbumin （OVA） respectively by the method of active ester, and the hybridoma cell lines secreting specific anti-E3 mAb were developed via monoclonal antidody technology. The dot immunoenzyme filtration assay in competitive-inhibition format was established with nitrocellulose membrane as solid-phase carrier and anti-E3 mAb as coating antibody. Results 5 hybridorna cell lines secreting specific anti-E3 mAb were obtained with E3-mAb titers in the range of 1 × 10^5 to 5 × 10^5 and the affinity constant （Ka） from 5. 1 ×10^8 L/mol to 5.2 × 10^9 L/mol. The limit of detecting （LOD） value for E3 was 2 ng/mL and the detection range was 2-200 ng/mL. Conclusion The dot immu- noenzyme filtration assay with high-speed detection, easy accessibility and high sensitivity was sucessfully established,

关 键 词：斑点免疫酶渗滤法 雌三醇 单克隆抗体 

分 类 号：R392.11[医药卫生—免疫学] R446.61[医药卫生—基础医学]