免疫荧光法检测杆状病毒滴度的建立及验证

Development and verification of indirect immunofluorescence assay for detection of baculovirus titer

目的 建立并优化生产用重组杆状病毒滴度间接免疫荧光检测方法,并进行初步验证.方法 将96孔板贴壁培养的SF9细胞,接种杆状病毒溶液共孵育一定时间后间接免疫荧光法显色.通过对细胞使用浓度、孵育时间及优化显色处理条件（固定液、封闭液、抗体种类及使用条件）,建立杆状病毒滴度间接免疫荧光检测方法,进行特异性、准确性、重复性和中间精密度验证,并与杆状病毒滴度酶联检测试剂盒检测结果进行比较.结果 细胞使用浓度为0.6×10^6个/ml、共孵育时间为3d;显色条件优化后确定了冰冻甲醛-丙酮溶液为最适固定溶液和5％的山羊血清封闭液,一抗和二抗稀释比例均为1：200、37℃温育1 h时显色效果最佳.用该方法检测不同样品,仅杆状病毒组出现特异性荧光斑点.本方法与试剂盒酶联法检测结果趋势相同,其线性相关系数R^2=0.996,表明两种方法具有良好的正相关性;重复性及中间精密度验证结果的变异系数CV均〈10％.结论 建立了重组杆状病毒滴度的间接免疫荧光检测方法,该方法具有良好的特异性、准确性、重复性和中间精密度,可用于生产中病毒滴度的检测.

Objective To develop, optimize and preliminarily verify an indirect immunofluores-cence assay （ IFA） for detecting the titer of recombinant baculovirus. Methods Conditions for performing IFA, including cell concentration, co-incubation time, reaction temperature, dilution ratio, reaction time and types of fixative solution, blocking liquid and antibodies, were optimized to establish an IFA method for the detection of baculovirus titer. Specificity, accuracy, reproducibility and intermediate precision of the es-tablished assay were verified. And the results were compared with those of baculovirus rapid titer kit. Re-sults The optimal cell concentration for coating was 0. 6×10^6 cell/ml, and the optimal reaction time be-tween viruses with cells was 3 d. The optimal conditions for conducting IFA were as follows： formaldehyde buffered acetone （-20℃） was used as fixative, normal goat serum was used as blocking liquid and the first and second generation antibodies at a dilution of 1 ： 200 were incubated at 37℃ for 1 h, respectively. Spe-cific fluorescence was observed only in baculovirus but not in others by using the method. No significant difference in virus titers was observed between the established IFA and baculovirus rapid titer kit. The two methods showed a good linear correlation （R2=0. 996）. Coefficients of variation for evaluating the reproduc-ibility and intermediate precision were less than 10%. Conclusion IFA for the detection of baculovirus ti-ter was established with good specificity, accuracy, reproducibility and intermediate precision.