BCSP31蛋白双抗夹心ELISA定量方法的建立及应用  

Establishment and application of double antibody sandwich ELISA quantitative method for BCSP31protein

作　　者：陈凯楠 李金斗 丁佳欣 冯嘉轩 于喜冰 单春晖 汪思捷 丁壮 CHEN Kainan;LI Jindou;DING Jiaxin;FENG Jiaxuan;YU Xibing;SHAN Chunhui;WANG Sijie;DING Zhuang(State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases,College of Veterinay Medicine,Jilin University,Changchun 130062,China;College of Clinical Medicine,Changchun University of Traditional Chinese Medicine,Changchun 130117,China)

机构地区：[1]吉林大学动物医学学院人畜共患传染病重症诊治全国重点实验室,吉林长春130062 [2]长春中医药大学临床医学院,吉林长春130117

出　　处：《中国兽医学报》2024年第4期699-705,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32072860);吉林省科技厅重点研发资助项目(20230202082NC);长春市科技局重点研发计划资助项目(21ZGN17)。

摘　　要：通过原核表达系统制备、纯化布鲁菌嵌合病毒样颗粒疫苗核心组分BCSP31蛋白,并将其作为免疫原及标准品分别免疫新西兰大白兔(1mg/只)和BALB/c鼠(200μg/只)制备兔源捕获多克隆抗体和鼠源检测多克隆抗体,对双抗夹心ELISA方法的捕获抗体和检测抗体最佳稀释倍数、封闭液种类及封闭条件、酶标二抗工作条件等参数进行优化,结果显示兔多克隆抗体最佳包被浓度和鼠多克隆抗体最佳稀释倍数均为1∶1600、3%脱脂奶粉封闭1h、酶标二抗最佳稀释倍数为1∶4000、酶标二抗孵育时间1h、显色时间为15min的条件下建立的双抗夹心ELISA方法效果最好。按照上述最佳反应条件对布鲁菌嵌合病毒样颗粒疫苗(BCSP31-cVLPs)核心抗原成分BCSP31蛋白进行定量检测,并对其敏感性、特异性、重复性等性能进行评价,结果显示1μL布鲁菌嵌合病毒样颗粒中的BCSP31含量约占总蛋白含量的14.187%;将布鲁菌嵌合病毒样颗粒稀释640倍后,P/N值仍大于2.1,有较高的敏感性;与FAdV、IBDV、NDV、AIV等其他病毒样颗粒均无交叉反应,特异性高;批间和批内变异系数均在10%以下,重复性良好。该方法的建立为推进布鲁菌嵌合病毒样颗粒疫苗的质量控制标准建立及商品化奠定了基础。In order to accurately quantify the effective content of the core component BCSP31protein of Brucella chimeric virus-like particle vaccine,a double-antibody sandwich ELISA method was established in this study.BCSP31was prepared and purified by prokaryotic expression system as immunogen and standard substance,rabbits and BALB/c mice were immunized to prepare rabbit-derived capture polyclonal antibody and mouse-derived detection polyclonal antibody.The parameters of the double antibody sandwich ELISA method,such as the optimal dilution times of capture antibody and detection antibody,the type of blocking solution and blocking conditions and the working conditions of enzyme-labeled secondary antibody were optimized.The results showed that the optimal coating concentration of rabbit polyclonal antibody and the optimal dilution ratio of mouse polyclonal antibody were 1∶1600,the optimal blocking condition was 3%skimmed milk powder for 1h,the optimal dilution ratio of enzyme-labeled secondary antibody was 1∶4000,the incubation time of enzyme-labeled secondary antibody was 1h,and the color development time was 15min.The core antigen component BCSP31protein of Brucella chimeric virus-like particle vaccine(BCSP31-cVLPS)was quantitatively detected under the above optimal reaction conditions,and its sensitivity,specificity and repeatability were evaluated.The results showed that the BCSP31 content in 1μL Brucella chimeric virus-like particles accounted for 14.187%of the total protein content.After diluting of Brucella chimeric virus-like particles 640times,the P/N value was still greater than 2.1,and there was no cross-reaction with other virus-like particles such as FAdV,IBDV,NDV and AIV.Specificity,inter-and intra-batch coefficients of variation were all below 10%,and repeatability was good.The establishment of this method lays a foundation for promoting the establishment of quality control standards and commercialization of Brucella chimeric virus-like particle vaccine.

关 键 词：布鲁菌病嵌合病毒样颗粒 BCSP31蛋白 双抗夹心ELISA方法 参数优化 应用 

分 类 号：S852.61[农业科学—基础兽医学] R535[农业科学—兽医学]