免疫单扩散法测定重组铜绿假单胞菌外毒素A蛋白含量方法的建立及其应用  

Establishment and application of a single immunodiffusion method for determining concentration of recombinant Pseudomonas aeruginosa exotoxin A

作　　者：李燕婷[1] 范锋锋[1] 冯宜扬[1] 金鑫[1] 吴朝今[1] 赵志强[1] 谢贵林[1] 谭小梅[1] 

机构地区：[1]兰州生物制品研究所有限责任公司,兰州730046

出　　处：《中国新药杂志》2015年第20期2346-2351,共6页Chinese Journal of New Drugs

基　　金：国家"重大新药创制"科技重大专项(2013ZX09402302-212)

摘　　要：目的:探索建立免疫单扩散法测定不同料液中重组铜绿假单胞菌外毒素A(recombinant Pseudomonas aeruginosa exotoxin A,r EPA)蛋白含量新方法。方法:对缓冲液系统、最佳抗体浓度和最佳观测时间等关键因素进行摸索,确定方法的最佳条件。在此基础上,对该方法的专属性、精密度、准确性和标准曲线的可靠性进行验证,并将该方法应用于r EPA柱层析纯化中不同样品目标蛋白含量的测定及回收率计算。结果:最佳缓冲系统为0.85%Na Cl溶液,最佳抗体用量为50μL·m L-1,最佳观测计算时间为4 h。方法具有较好的专属性、精密度、准确性和线性。结论:初步建立了免疫单扩散试验测定不同料液中r EPA蛋白含量新方法,为其纯化工艺的优化奠定了基础。Objective： To establish a single immunodiffusion method for determining concentration of recombinant Pseudomonas aeruginosa exotoxin A（ r EPA） in different solutions. Methods： The key factors,such as buffer system,antibody volume and observation time,were explored,and the optimum conditions of the method were determined. Then,the specificity,precision,accuracy and reliability of standard curve was validated. Meantime,the method was applied to determine the target protein content,and to calculate recovery rate of r EPA column chromatography purification. Results： The optimum buffer system was 0. 85% Na Cl solution,the optimum antibody dose was 50 μL·m L- 1,and the optimum observation time was 4 h. The method had good specificity,precision,accuracy and linearity. Conclusion： A single immunodiffusion method for determining the r EPA protein content in different materials has been established preliminarily,and it lays the foundation for the optimization of the purification process.

关 键 词：免疫单扩散法 方法验证 重组铜绿假单胞菌外毒素A 蛋白质纯化 收率 

分 类 号：R915[医药卫生—微生物与生化药学]