摘要
目的应用细胞荧光转化灶（fluorescence focus units assay,FFU）法检测狂犬病病毒滴度。方法将狂犬病病毒稀释后接种BSR细胞,培养22~24 h,用FITC标记的抗狂犬病病毒特异性抗体进行染色,计数病毒感染细胞的荧光灶数,计算病毒滴度。采用乳鼠半数致死量（median lethal dose,LD50）法和建立的方法分别测定14批狂犬病病毒滴度,分析两者间的相关性;取同一份病毒液,于不同时间点检测3次,每个时间点重复检测6次,验证方法的重复性;取3份滴度不同的病毒液,由2个操作者在不同时间重复测定3次,验证方法的中间精密性;将同一份病毒液按2倍梯度稀释,共9个稀释度,分别检测病毒滴度,重复测定3次,确定该方法的线性范围。应用建立的方法检测38批CTNCEC株狂犬病病毒滴度。结果乳鼠LD50法测定14批狂犬病病毒的-Lg LD50值范围为4.20~8.19,FFU法Lg FFU值范围为3.76~7.69,两种方法检测结果趋势相同,线性回归相关系数R-Sq（调整）=96.1%,存在良好的正相关性;同一个时间点检测6次及不同时间点检测3次的CV值均小于2.00%;两个操作者在不同时间重复测定3次的CV值均小于8.00%,两个操作者之间的检测结果差异无统计学意义（P〉0.05）;样品浓度与Lg FFU呈良好的线性关系,R-Sq（调整）=99.3%,线性范围为2.26~6.12。38批CTNCEC株狂犬病病毒滴度平均Lg FFU在3.63~7.69之间,SD值在0.021~0.57之间,CV值小于10.00%。结论建立的细胞FFU法准确性、重复性、中间精密度好,该方法可用于检测狂犬病病毒滴度。
Objective To determine the titer of rabies virus by cell fluorescence focus unit（FFU） assay. Methods Rabies virus was diluted, inoculated to BSR cells, cultured for 22 ~ 24 h, and stained with FITC-labeled specific antibody against rabies virus to count the fluorescence foci in infected cells, based on which the virus titer was calculated. The titers of 14 batches of rabies virus were determined by median lethal dose（LD50）assay in suckling mice and the developed method respectively, and the relationship between the two methods was analyzed. The same virus sample was determined at 3 time points, 6 times for each, to verify the reproducibility of the method. Three virus samples at different titers were determined for 3 times at various time points by 2 staff separately to verify the intermediate precision of the method. The same virus sample was 2-fold serially diluted to 9 dilutions, each of which was tested for titer for 3 times to determine the linear range of the method. A total of 38 batches of rabies virus CTNCEC strain were determined for titer by the developed method. Results The range of-Lg LD50 values of 14 batches of rabies virus determined by LD50 assay in suckling mice was 4. 20 ~ 8. 19, while the range of Lg FFU by FFU assay was 3. 76 ~ 7. 69. The-Lg LD50 and Lg FFU exhibited the same trends, which showed a significantly linear correlation（adjusted R2 = 96. 1）. The CVs of samples determined for 6 times at the same time point and for 3 times at various time points were less than 2. 00%. However, the CVs of samples determined for 3 times at various time points by two staff were less than 8. 00%, which showed no significant difference（P 〉 0. 05）. The sample concentration showed a good linear relationship to Lg FFU, with an adjusted R2 value of 99. 3%,of which the linear range was 2. 26 ~ 6. 12. The mean Lg FFU of 38 batches of CTNCEC strain was 3. 63 ~ 7. 69, with a standard deviation（SD）of 0. 021 ~ 0. 57 and a CV value of less than 10. 00%. Conclusion The developed FFU assay showed high accuracy, reproducibility and intermediate precision, which might be used for the determination of rabies virus titer.
出处
《中国生物制品学杂志》
CAS
CSCD
2015年第10期1092-1096,共5页
Chinese Journal of Biologicals
