GRP78单抗多抗的制备鉴定及其免疫学检测方法的建立  

Preparation and identification of GRP78 monoclonal and polyclonal antibodies and establishment of an immunological detection method

作　　者：阳媛[1] 夏露露 师悦嫄 魏杰[1] 汪德强 牛司强[3] Yang Yuan;Xia Lulu;Shi Yueyuan;Wei Jie;Wang Deqian;Niu Siqiang(Key Laboratory of Clinical Laboratory Diagnosis,College of Laboratory Medicine,Chongqing Medical University;Department of Clinical Laboratory,The First Affiliated Hospital of Chongqing Medical University;Key Laboratory of Molecular Biology on Infectious Diseases Established by Ministry of Education)

机构地区：[1]重庆医科大学检验医学院临床检验诊断重点实验室,重庆400016 [2]重庆医科大学附属第一医院检验科,重庆400016 [3]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《重庆医科大学学报》2020年第6期817-823,共7页Journal of Chongqing Medical University

基　　金：重庆市教委科学技术研究资助项目(编号:KJ1702022);重庆市卫计委2016年医学科研面上资助项目(编号:2016MSX001)。

摘　　要：目的:制备与鉴定抗GRP78兔多克隆抗体与腹水型单克隆抗体,进一步建立可检测GRP78蛋白的双抗夹心ELISA方法。方法:利用课题组前期已经成功构建的pW28-GRP78重组质粒,IPTG诱导表达后经过Ni2+-NTA亲和层析柱分离纯化获得高纯度GRP78重组蛋白;获得的GRP78蛋白免疫新西兰兔制备多克隆抗体,辛酸-硫酸铵沉淀后DEAE离子交换层析法纯化;制备GRP78腹水型单克隆抗体并鉴定其亚型,Protein G亲合柱纯化;采用Western blot、免疫荧光、免疫组化等方法对获得的GRP78兔多抗与鼠单抗进行鉴定;建立一个可检测GRP78蛋白的双抗夹心ELISA免疫学方法。结果:高效获得高纯度GRP78重组蛋白,成功制备兔多抗,同时获取8株腹水型单克隆抗体,选择效价最高的McAb-1用于后续双抗夹心ELISA实验,McAb-1为G2a型;进一步的鉴定结果表明制备的抗体皆具有良好免疫反应性及特异性;单抗作为捕获抗体,多抗作为检测抗体,HRP标记的羊抗兔酶标二抗,成功建立可检测人GRP78的双抗夹心ELISA方法,检测下限为152.3 ng/mL。结论:成功获取了高效价、高灵敏度及高特异性的GRP78兔多抗及腹水型鼠单抗,并在此基础上建立了可检测人GRP78蛋白的双抗体夹心ELISA检测系统,为GRP78的进一步研究奠定重要基础,具有良好的临床应用前景。Objective:To investigate the preparation and identification of anti-GRP78 rabbit polyclonal antibodies and ascites monoclonal antibodies,and to establish a method of double-antibody sandwich enzyme-linked immunosorbent assay(ELISA)for the detection of GRP78 protein.Methods:The recombinant plasmid pW28-GRP78 that was successfully constructed by previous research was used,and after expression induction by IPTG,the Ni2+-NTA affinity chromatography column was used to obtain high-purity GRP78 recombinant protein.New Zealand rabbits were immunized with the GRP78 protein obtained to prepare polyclonal antibody,which was purified by DEAE ion-exchange chromatography after caprylic acid-ammonium sulfate precipitation.GRP78 ascites monoclonal antibody was prepared and its subtypes were identified,and it was purified by the Protein G affinity column.Related methods such as WB,IF,and IHC were used to identify the GRP78 rabbit polyclonal antibody and rat monoclonal antibody;a double-antibody sandwich ELISA method was established for the detection of GRP78 protein.Results:High-purity GRP78 recombinant protein was obtained efficiently,and rabbit polyclonal antibody was successfully prepared.A total of 8 ascites monoclonal antibodies were obtained,among which McAb-1 with the highest titer was selected for the subsequent double-antibody sandwich ELISA and was identified as G2 a type.Further identification showed that all the antibodies prepared had good immunoreactivity and specificity.With the monoclonal antibody as the capture antibody and the polyclonal antibody as the detection antibody,the HRP-labeled goat antirabbit secondary antibody was used to successfully establish a double-antibody sandwich ELISA for the detection of human GRP78,with a lower limit of detection of 152.3 ng/mL.Conclusion:GRP78 rabbit polyclonal antibody and rat ascites monoclonal antibody with high titer and sensitivity are successfully obtained,and a double-antibody sandwich ELISA system is successfully established for the detection of human GRP78 protei

关 键 词：GRP78 多克隆抗体 腹水型单克隆抗体 鉴定 双抗夹心ELISA 

分 类 号：R392.ll[医药卫生—免疫学]