MPT64抗体的适体在结核病血清学检测中的应用  

The research of MPT64 antibodies aptamer of Mycobacterium tuberculosis in the serological diagnosis

作　　者：蔡江丽[1] 秦莲花[2] 刘忠华[2] 王洁[2] 胡忠义[2] 

机构地区：[1]苏州大学基础医学与生物科学学院,215123 [2]同济大学附属上海市肺科医院,上海市结核病(肺)重点实验室,200433

出　　处：《中华微生物学和免疫学杂志》2010年第2期180-184,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家科技重大专项（2008ZX10003003-02）;上海市科委项目（08DZ2291500,08DZ2271300）;上海市公卫重点学科项目（08GWZX0104）

摘　　要：目的 利用SELEX技术筛选MPT64抗体的适体建立混合夹心ELISA检测体系,应用于临床血清标本的检测,探讨该方法 潜在的实验室诊断价值.方法 利用竞争ELISA检测方法 ,测定不同浓度的MPT64抗原对最后一轮ssDNA文库与靶物质亲和力的抑制率,选取优势序列且亲和性值较高的适体用于检测,优化混合夹心ELISA检测方法 ,确定MPT64抗体的检测下限和线性范围,同时检测230例临床血清标本.结果 在竞争ELISA结果 中,总反应体系为100μl,MPT64抗原浓度由2μg/ml增加到256 μg/ml时,对ssDNA文库的抑制率也由0.25%增加到80%.优化的混合夹心ELISA的检测体系：ssDNA包被浓度为0.1μg/孔、血清稀释度为1/200、辣根过氧化物酶标记的羊抗人IgG抗体浓度为1/40 000.MPT64抗体的检测下限为3 mg/L,线性范围为10～1000 mg/L.利用该体系检测100例结核病患者、100例健康体检者和30例非结核病患者血清,检测结果 用GraphpadPrism软件进行分析,结核病组与健康体检组、结核病组与非结核疾病对照组差异均具有统计学意义（P〈0.001）.统计学分析得到检测的特异性与敏感性分别为96.1%和31.0%.结论 利用适体建立的混合夹心ELISA用于结核病的血清学诊断具有一定的诊断价值.Objective To establish mixed-sandwich ELISA detection system by screening aptam-ers of MPT64 antibodies with SELEX to detect clinical serum samples, and explore the potential laboratory diagnosis value of this method. Methods To detect the affinity of the final round ssDNA library to MPT64 antibodies inhibited by MPT64 antigen with the competitive ELISA method, optimize the mixed-sandwich ELISA detection method that was aptamer-serum-horseradish peroxidase labeled goat anti-human IgG anti-body detection system to detect 230 cases of clinical serum samples as well as the lowest concentration of MPT64 antibodies and the linear range. Results In competitive ELISA test results, the percentage of inhi-bition effect of MPT64 antigen to final round ssDNA library is from 0.25% to 80% when the MPT64 antigen concentration rised from 2 μg/ml to 256 μg/ml. The Optimized detection system of mixed-sandwich ELISA was constitute of the concentration of ssDNA coated with 0.1μg/hole, serum dilution of 1/200, horseradish peroxidase labeled goat anti-human IgG antibody concentration of 1/40 000. The lowest concentration of MPT64 antibody is 3 mg/L and the linear range is between 10 mg/L and 1000 mg/L. The serum samples of 100 cases of tuberculosis patients, 100 healthy individuals and 30 cases of non-tuberculesis were tested in this system and the test result was analyzed with Graphpad Prism, the difference of tuberculosis group and healthy group was statistically significant （P〈0.001 ）, the difference of TB group and non-TB control group was also statistically significant （P〈0.001）. The specificity and the sensitivity was 96.1% and 31.0% re-spectively. Conclusion The aptamer mixed-sandwich ELISA method will play an important role in the sero-logical diagnosis of tuberculosis.

关 键 词：MPT64抗体 SELEX 适体 SSDNA 血清学检测 

分 类 号：R52[医药卫生—内科学] R446.6[医药卫生—临床医学]