结核DNA疫苗宿主蛋白残留检测方法的建立

Establishment of Detection Method for Host Protein Residues in Tuberculosis DNA Vaccine

目的建立结核DNA疫苗中大肠杆菌宿主蛋白残留量检测的方法。方法采用双抗夹心酶联免疫吸附法(ELISA法)建立结核DNA疫苗中宿主蛋白残留量检测的方法,对方法的准确度、专属性、线性范围和耐用性等进行验证,并采用建立的方法对结核DNA疫苗原液进行测定。结果大肠杆菌宿主蛋白含量在2~24ng·L^(-1)范围内,相关系数R 2>0.990,线性关系良好;该法宿主蛋白含量在高中低浓度范围内,回收率在70%~120%之内,准确度良好;该法不易受辅料、核酸、孵育温度和孵育时间影响,专属性和耐用性较好;三批原液的宿主蛋白残留量(HCP)均<1μg·mg^(-1)。结论此方法有较好的准确度、专属性、重复性、中间精密度和耐用性,可用于结核DNA疫苗的宿主蛋白残留量的检测。

OBJECTIVE To develop a detection method for the residual content of E.coli host protein in tuberculosis DNA vaccine.METHODS The residual content of host protein of tuberculosis DNA vaccine was detected by double antibody sandwich enzyme-linked immunosorbent assay(ELISA),and the accuracy,specificity,linear range and durability of the method were verified.The established method was used to test the TB DNA vaccine stock solution.RESULTS The linear relationship was good which that the correlation coefficient R 2 was>0.990 and the content of E.coli host protein was in the range of 2-24 ng·L^(-1).The accuracy recovery was 70%-120%with the content of host protein was in the range of high,middle and low concentrations.The method not only has strong specificity which that is less affected by excipients and nucleic acids,but also the durability is good which that is less affected by changing the incubation temperature and incubation time.The residual amount of host protein(HCP)of three batches of stock solution is less than 1μg·mg^(-1).CONCLUSION This method has good accuracy,specificity,repeatability,intermediate precision and durability,and can be used to determine the residual content of host protein in Tuberculosis DNA vaccine.