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 BCSP31 protein
陈凯楠 1李金斗 1丁佳欣 1冯嘉轩 2于喜冰 1单春晖 1汪思捷 1丁壮1
作者信息
- 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
- 折叠
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(1 mg/只)和BALB/c鼠(200 μg/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∶1 600、3％脱脂奶粉封闭1 h、酶标二抗最佳稀释倍数为1∶4 000、酶标二抗孵育时间1 h、显色时间为15 min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、IB-DV、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.
Abstract
In order to accurately quantify the effective content of the core component BCSP31 pro-tein of Brucella chimeric virus-like particle vaccine,a double-antibody sandwich ELISA method was established in this study.BCSP31 was prepared and purified by prokaryotic expression system as immunogen and standard substance,rabbits and BALB/c mice were immunized to prepare rab-bit-derived capture polyclonal antibody and mouse-derived detection polyclonal antibody.The pa-rameters 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∶1 600,the optimal blocking condition was 3％skimmed milk powder for 1 h,the optimal dilution ratio of enzyme-labeled secondary antibody was 1∶4 000,the incubation time of enzyme-labeled secondary antibody was 1 h,and the color development time was 15 min.The core antigen component BCSP31 protein of Brucella chimeric virus-like particle vac-cine(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 1 μL Brucella chimeric virus-like particles accounted for 14.187％of the total protein content.After diluting of Brucella chimeric virus-like particles 640 times,the P/N value was still greater than 2.1,and there was no cross-reaction with other virus-like particles such as FAdV,IB-DV,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Key words
Brucella chimeric virus-like particles/BCSP31 protein/double antibody sandwich ELISA method/parameter optimization/application引用本文复制引用
基金项目
国家自然科学基金(32072860)
吉林省科技厅重点研发项目(20230202082NC)
长春市科技局重点研发计划(21ZGN17)
出版年
2024
NETL
NSTL
万方数据