结核分枝杆菌MPT64双抗体夹心ELISA检测方法的建立及初步应用  

Establishment and preliminary application of Mycobacterium tuberculosis MPT64 double-antibody sandwichELISA

作　　者：谢燕玲 宁唤唤 路延之 康健 白鹭 代婷 胡家豪 徐子晴 刘博 柏银兰 XIE Yanling;NING Huanhuan;LU Yanzhi;KANG Jian;BAI Lu;DAI Ting;HU Jiahao;XU Ziqing;LIU Bo;BAI Yinlan(School of Life Sciences,Yan'an University,Yan'an 716000,China;Department of Microbiology and Pathogen Biology,School of Basic Medicine,Air Force Medical University;Military Medical Innovation Center,Air Force Medical University;Cadets of the 5th Brigade,School of Basic Medicine,Air ForceMedical University)

机构地区：[1]延安大学生命科学学院,陕西延安716000 [2]空军军医大学基础医学院微生物与病原生物学教研室 [3]空军军医大学军事医学创新中心 [4]空军军医大学基础医学院学员第五大队

出　　处：《中国病原生物学杂志》2023年第11期1284-1290,共7页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81971560,82272343);国家“十三五”重大传染病专项课题(No.2018ZX10302302002004);陕西省重点课题(No.2022ZDLSF01-07)。

摘　　要：目的建立结核分枝杆菌(Mycobacterium tuberculosis,Mtb)分泌蛋白MPT64的双抗体夹心ELISA检测方法并评价初步应用效果。方法采用间接ELISA和ELISA叠加试验在各株抗MPT64 mAb中筛选出亲和力较高的识别不同抗原表位的配对抗体;Protein A亲和层析纯化配对的2株mAb腹水,其中1株mAb偶联生物素(Biotin)作为检测抗体,另1株作为捕获抗体,棋盘滴定法优化捕获抗体和检测抗体的工作浓度,建立双抗体夹心ELISA方法;以常规条件作为初始反应条件,依次优化包被、封闭、抗原、抗体以及底物反应环节的条件;以MPT64抗原检测评估建立的ELISA方法的检测限、线性范围、精确性、准确性和特异性;采用该方法检测Mtb H37Ra不同阶段培养上清中MPT64含量,初步评价应用效果。结果ELISA法成功筛选出配对抗体H2F4和A5B2 mAb;液相色谱法从腹水中纯化获得的mAb滴度分别为1∶102400、1∶51200。以生物素偶联的A5B2 mAb作为检测抗体,以H2F4 mAb为捕获抗体,建立并优化MPT64双抗体夹心ELISA方法,确定的试验条件为:1.25μg/mL H2F4 mAb包被酶标板,4℃静置过夜,洗涤;2%BSA室温封闭1 h,洗涤;加入待测样品室温孵育2 h,洗涤;加入2.5μg/mL Biotin-A5B2 mAb室温孵育2 h,洗涤;加入0.5μg/mL Avidin-HRP室温孵育1 h,洗涤;加入TMB显色检测。该ELISA方法对MPT64蛋白的检测下限为39 ng/mL,线性范围为39~1250 ng/mL,具有良好的特异性;该方法检测Mtb培养物,第12 d培养上清呈阳性反应。结论建立的Mtb MPT64双抗体夹心ELISA方法精确性、准确性,特异性良好,可用于MPT64定量检测和Mtb生长检测,为TB快速诊断试剂的研制提供了实验依据。Objective In order to establish a double-antibody sandwich ELISA for Mycobacterium tuberculosis(Mtb)secreted protein MPT64,and its preliminary application was explored.M.ethodsIndirect ELISA and ELISA double antibody binding system were used to screen paired antibodies with high affinity and recognition of different epitopes from several anti-MPT64 mAbs.Protein A affinity chromatography purified paired two ascites mAbs.One of the two mAbs was conjugated with biotin and used as the detection antibody,and another mAb was used as the capture antibody.The checkerboard titration was used to optimize the concentration of capture antibody and detection antibody,and the double antibody sandwich ELISA was established.The conventional conditions were used as the initial reaction conditions,and the conditions of coating,blocking,antigen-antibody and substrate reaction links were optimized gradually.MPT64 antigen detection was used to evaluate the key parameters such as detection limit,linear range,precision,accuracy and specificity of the ELISA system established by this institute.The system was applied to analyse the content of MPT64 in the culture supernatant at different stages of Mtb H37Ra growth.Results The paired antibodies of anti-MPT64 H2F4 and A5B2 mAb were successfully screened by ELISA.The purified mAbs were obtained by liquid chromatography.The antibody titers of the mAbs were 1:102400 and 1:51200.The MPT64 double antibody sandwich ELISA method was established,with biotin-coupled A5B2 mAb as detection antibody and H2F4 mAb as trapping antibody.The conditions of ELISA method were optimized as follows,1.25μg/mL H2F4 mAb coated enzyme plate,4 C overnight,washed,2%BSA blocked at room temperature for 1 h,washed,samples were incubated for 2 h at room temperature,washed,2.5μg/mL Biotin-A5B2 mAb incubation at room temperature for 2 h,wash,0.5μg/mL Avidin-HRP incubation at room temperature for 1 h,wash,TMB chromogenic detection.The lower limit of detection of MPT64 of ELISA system was 39 ng/mL,the linear range was 39-

关 键 词：结核分枝杆菌 分泌抗原 MPT64 单克隆抗体 双抗体夹心ELISA 

分 类 号：R378.91[医药卫生—病原生物学]