Laboratory of Mycobacterial Diseases and Cellular Immunology, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD 20892, USA.
J Microbiol Methods. 2012 Sep;90(3):245-9. doi: 10.1016/j.mimet.2012.05.015. Epub 2012 May 28.
The viability of BCG vaccine has traditionally been monitored using a colony-forming unit (CFU) assay. Despite its widespread use, results from the CFU assay can be highly variable because of the characteristic clumping of mycobacteria, their requirement for complex growth media, and the three week incubation period needed to cultivate slow-growing mycobacteria. In this study, we evaluated whether an ATP luminescence assay (which measures intracellular ATP content) could be used to rapidly estimate the viability of lyophilized and/or frozen preparations of six different BCG vaccine preparations - Danish, Tokyo, Russia, Brazil, Tice, and Pasteur - and two live attenuated mycobacterial vaccine candidates - a ΔlysAΔpanCD M. tuberculosis strain and a ΔmmaA4 BCG vaccine mutant. For every vaccine tested, a significant correlation was observed between intracellular ATP concentrations and the number of viable attenuated bacilli. However, the extractable intracellular ATP levels detected per cell among the different live vaccines varied suggesting that validated ATP luminescence assays with specific appropriate standards must be developed for each individual live attenuated vaccine preparation. Overall, these data indicate that the ATP luminescence assay is a rapid, sensitive, and reliable alternative method for quantifying the viability of varying live attenuated mycobacterial vaccine preparations.
BCG 疫苗的存活率传统上是通过集落形成单位（CFU）测定法来监测的。尽管该方法应用广泛，但由于分枝杆菌的特征性聚集、其对复杂生长培养基的要求以及培养生长缓慢的分枝杆菌所需的三周孵育期，CFU 测定法的结果可能存在很大差异。在这项研究中，我们评估了是否可以使用 ATP 发光测定法（测量细胞内 ATP 含量）来快速估计六种不同 BCG 疫苗制剂（丹麦、东京、俄罗斯、巴西、Tice 和 Pasteur）以及两种减毒活分枝杆菌疫苗候选物（lysAΔpanCD M. tuberculosis 株和 mmaA4 BCG 疫苗突变株）的冻干和/或冷冻制剂的存活率。对于测试的每种疫苗，都观察到细胞内 ATP 浓度与减毒活细菌的数量之间存在显著相关性。然而，不同活疫苗中每个细胞检测到的可提取细胞内 ATP 水平存在差异，这表明必须针对每种单独的减毒活疫苗制剂开发经过验证的、具有特定合适标准的 ATP 发光测定法。总体而言，这些数据表明，ATP 发光测定法是一种快速、敏感和可靠的替代方法，可用于定量检测不同减毒活分枝杆菌疫苗制剂的存活率。
