Pyke Alyssa T, Phillips Debra A, Chuan Teck F, Smith Greg A
Public Health Virology, Queensland Health Scientific Services, Coopers Plains, Australia.
BMC Microbiol. 2004 Jan 14;4:3. doi: 10.1186/1471-2180-4-3.
Sucrose density gradient centrifugation and cross-flow filtration methods have been developed and standardised for the safe and reproducible production of inactivated arbovirus antigens which are appropriate for use in diagnostic serological applications.
To optimise the maximum titre of growth during the propagation of arboviruses, the multiplicity of infection and choice of cell line were investigated using stocks of Ross River virus and Barmah Forest virus grown in both mosquito and mammalian cell lines. To standardise and improve the efficacy of the inactivation of arboviral suspensions, stocks of Ross River virus, Barmah Forest virus, Japanese encephalitis virus, Murray Valley encephalitis virus and Alfuy virus were chemically inactivated using binary ethylenimine at a final concentration of 3 mM. Aliquots were then taken at hourly intervals and crude inactivation rates were determined for each virus using a plaque assay. To ensure complete inactivation, the same aliquots were each passaged 3 times in Aedes albopictus C6/36 cells and the presence of viral growth was detected using an immunofluorescent assay. For larger quantities of viral suspensions, centrifugation on an isopycnic sucrose density gradient or cross-flow filtration was used to produce concentrated, pure antigens or partially concentrated, semi-purified antigens respectively.
The results of the propagation experiments suggested that the maximum viral titres obtained for both Ross River virus and Barmah Forest virus were affected by the incubation period and choice of cell line, rather than the use of different multiplicity of infection values. Results of the binary ethylenimine inactivation trial suggested that standardised periods of 5 or 8 hours would be suitable to ensure effective and complete inactivation for a number of different arboviral antigens.
Two methods used to prepare inactivated arbovirus antigens have been standardised to minimise production failure and expenditure and to provide reagents that conform to the highest quality and safety requirements of a diagnostic serology laboratory. The antigens are suitable for use in either enzyme linked immunosorbent assays or haemagglutination inhibition assays and the optimised protocols can be directly applied to produce antigens from new or emerging arboviral pathogens.
已开发并标准化了蔗糖密度梯度离心法和错流过滤法，用于安全且可重复地生产适用于诊断血清学应用的灭活虫媒病毒抗原。
为了优化虫媒病毒增殖过程中的最大生长滴度，使用在蚊子和哺乳动物细胞系中培养的罗斯河病毒和巴马森林病毒毒株，研究了感染复数和细胞系的选择。为了标准化并提高虫媒病毒悬液的灭活效果，使用终浓度为3 mM的双乙烯亚胺对罗斯河病毒、巴马森林病毒、日本脑炎病毒、墨累谷脑炎病毒和阿尔富伊病毒毒株进行化学灭活。然后每隔一小时取等分试样，使用蚀斑试验确定每种病毒的粗灭活率。为确保完全灭活，将相同的等分试样分别在白纹伊蚊C6/36细胞中传代3次，并使用免疫荧光试验检测病毒生长情况。对于大量病毒悬液，分别使用等密度蔗糖密度梯度离心或错流过滤来生产浓缩的纯抗原或部分浓缩的半纯化抗原。
增殖实验结果表明，罗斯河病毒和巴马森林病毒获得的最大病毒滴度受潜伏期和细胞系选择的影响，而非不同感染复数的使用。双乙烯亚胺灭活试验结果表明，5或8小时的标准化时间适用于确保多种不同虫媒病毒抗原的有效和完全灭活。
已对用于制备灭活虫媒病毒抗原的两种方法进行了标准化，以尽量减少生产失败和成本，并提供符合诊断血清学实验室最高质量和安全要求的试剂。这些抗原适用于酶联免疫吸附试验或血凝抑制试验，优化后的方案可直接应用于从新出现的虫媒病毒病原体生产抗原。
