Sucrose density gradient centrifugation and cross-flow filtration methods for the production of arbovirus antigens inactivated by binary ethylenimine

摘要

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.