For Preparing The Antigen Of Rabies Immunoglobuline With Vero Cells

Currently, the human-used rabies vaccine is generally used as antigen to generate horse anti-rabies virus antibody. The human-used rabies vaccine is routinely prepared from virus-infected cells such as hamster kidney cells or Vero cells other than brain tissue culture. Because the human-used vaccine contains human albumin as a protecting agent, which increases the production cost and causes the non-specific immune response, it cannot be directly applied for horse antibody production. To solve the above potential problems, in this study,we investigate the preparation of the antigen specifically used for horse antibody production. First, Vero cells and rabies viruses (aG strain) were adapted to the culture medium containing horse serum, the adapted cells and viruses contain higher stability and better antibody quality. Next, we studied the cell culture conditions for large-scale production. The optimized condition was determined, to generates the horse-used antigen, excellent antigenicity, high homogenous property, no human components, and high reliability.Our results showed that Vero cells could adapt to the horse-serum culture medium in a short time, taking only three generations. There is no significant difference in the Vero cell growth curve between horse serum medium and fetal bovine serum. To obtain the high titer of rabies virus, we further adapted the virus culture in horse serum medium by infecting Vero cells for 18 generations. The resulting virus titer reached to the level of 7.01ogLD50/mL. The comprehensive quality control tests demonstrated that the horse-serum adapted Vero cells and rabies virus have met the requirements for antigen production, in accordance with "the Chinese Requirements for Biologics Year 2000"Our results shows that the best range of the infection index is 0.001-0.1 M.O.I. The resulting virus titer could reach up to 6.5 logLD₅₀/mL. The best concentration of the horse serum was 1%-3%, which could enhance the virus titer up to 7.17 logLD₅₀/mL within 5-8 days, and then sustained the virus titers at 6.51ogLD₅₀/mL forabout 11 days. Our results demonstrated that the virus growth reached the peak between 5 to 8 days, and virus replication could last for over 20 days.We also found that the PH was dropped dramatically in the late stages of virus culture, and the re-titration of PH to 7.6 on day 3 could significantly improve the virus titers by day 5 and day 8.In combination of our results, the optimal culture condition is: seed virus titer of 7.51ogLDso/mL, 0.01M.O.I, 1% of horse serum, 35°C culture temperature, and adjust PH on day 3. Based on the above condition, three batches of rabies virus antigen were generated through the procedure including virus collection, concentration, inactivation, and centrifugation. Quality control tests indicated they have met the required indexes according "the Chinese Requirements for Biologies Year 2000" and showing the potency of 6.0IU/mL for each of batches.In summary, we have optimized rabies virus antigen preparation procedure specifically for horse anti-rabies antibody production. With the optimized techniques, all the conditions such as production cycle, productivity, quality control and the production cost are similar to those of using fetal bovine serum, except horse serum was used to replace the human albumin. Using the antigen from such protocol, there will be no need to remove anti-human albumin antibody, so that this procedure can significantly reduce the cost and increase the recovery of anti-rabies virus antibody, therefore, the improved procedure will have a wide application in horse anti-rabies virus antibody production.