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目的对冻干甲肝减毒活疫苗成品检定中关键性项目的检定方法进行优化。方法(1)感染性滴度检测:建立微量免疫荧光法,并与常规组织培养ELISA法比较;(2)牛血清白蛋白残留量检测:比较反向间接血凝法、酶联免疫法的重复性、精确性;(3)水分含量测定:建立助溶剂加入法,并比较助溶剂加入前后冻干疫苗水分含量检测结果。结果(1)微量免疫荧光法灵敏度与常规组织培养ELISA法相仿(P>0.05),重复性佳,可提前1周左右出结果;可用Leica Qwin荧光定量分析系统标定化判断结果。(2)反向间接血凝法测定结果不稳定,2批疫苗5次测定结果的变异系数(CV)为39.12%,34.23%;酶联免疫法的重复性,精确性均较佳(CV)为2.12%,2.82%,回收率为101.73%。(3)助溶剂加入前后2批疫苗5次测定结果的CV分别由8.32%,9.03%降为1.85%,1.82%。结论微量免疫荧光法有望代替常规组织培养ELISA法用于疫苗滴度检测;牛血清白蛋白残留量测定中酶联免疫法优于反向间接血凝法;加入助溶剂有利于提高疫苗水分含量检测的重复性。
OBJECTIVE To optimize the assay of the key items in the freeze-dried hepatitis A attenuated live vaccine product testing. Methods (1) Infectious titer test: The establishment of micro-immunofluorescence method, and compared with conventional tissue culture ELISA; (2) Determination of bovine serum albumin: reverse indirect hemagglutination, enzyme-linked immunosorbent repeat Sex, accuracy; (3) determination of moisture content: the establishment of co-solvent addition method, and compared with the co-solvent before and after the freeze-dried vaccine moisture content test results. Results (1) The sensitivity of the MFA method was similar to that of the conventional tissue culture ELISA (P> 0.05). The reproducibility was good and the results could be obtained about 1 week in advance. The results of the system calibration could be analyzed by Leica Qwin fluorescence quantitative analysis system. (2) The results of reverse indirect hemagglutination assay were unstable. The coefficient of variation (CV) of 5 batches of two batches of vaccine were 39.12% and 34.23%, respectively. The reproducibility and accuracy of enzyme-linked immunosorbent assay (ELISA) 2.12%, 2.82%, the recovery rate was 101.73%. (3) Before and after the co-solvent was added, the CVs of the five batches of vaccines decreased from 8.32% and 9.03% to 1.85% and 1.82%, respectively. Conclusion Micro-immunofluorescence assay is expected to replace routine tissue culture ELISA for vaccine titer detection; enzyme-linked immunosorbent assay (ELISA) is superior to reverse indirect hemagglutination assay in the determination of bovine serum albumin residues; Repeatability.