Production Process Optimization For Hog Cholera Lapinized Vaccine And Application Of ELISA Method In Quantitative Determination Of Antigens

Take CSFV attenuated vaccine strain of Chinese rabbit (CSFV C Strain) as experimental materials to study the basic features and laws of the strain's proliferation in primary bovine testicular cell. First we used conventional cells real-time counting method and settled proliferation curves of calves'testicular cells and infected cells, finding that with CSFV calves testicular cells not only did not produce cytopathic cells, but promoted cell division proliferation and the growth number of cells in 48 hours and number of infected calves testicular cells'division proliferation were as 127.2 percent as uninfected cells. We established a conventional virus proliferation curve by the way of direct immunofluorescent method (DIF) to screen out the toxin production model for choosing greatest cell density, most volume and best way of inoculation.Based on the established cell and virus growth curves and toxin production model, and according to the actual conditions of GMP workshop, we further work from production of toxic species breed, preparation of primary cell, infection of CSFV, regulation of maintenance media after CSFV infection, repeatedly generation of CSFV bovine testicular cells and other factors effecting on proliferation of CSFV.Strictly control and high quality of production of toxic species are necessary guarantee for standard content of virus vaccine.In the course of preparation of primary cell, according to"People's Republic of China Veterinary Biological Products Rules" (hereinafter referred to as: "Rules"), bovine testis are gathered sterilely. And based on the guidance of"Rules"adjust the digestion time and temperature to simultaneously reach the double effect of increasing cell number and improving cell quality. We adjusted the digestion time regulated in"Rule"to that after 30 minutes'0.25% trypsin's digestion in the temperature of 37℃the digestion would continue in lower temperature. As a result, per 1g testis can be prepared into 400-500ml of 3×105 ml cell suspension, which is as twice as traditional digestion does, and the cell viability increases significantly. Lowering the temperature of the post-digestion can reduce the strength of pancreatin's role to lessen injury on digested cell.Spleen tissue toxic has a strong stimulating effect on sub-bovine testicular cells, so we should reduce the toxic in the course of production of inoculation. Simultaneously, add some cell venom whose virus content is more than 106 to relieve the stimulating effect, reduce the de-bottle rate of cells and improve the harvest of virus solution.During the harvest of virus solution, increase the amount of maintenance medium, that is adjusting it from 10% regulated in"Rules"to 20%, and extend the harvest time from 4 days to 8 days one time. After the test in rabbits, the virus content could still be 5×104ml.In terms of virus content determination, we applied CSFV antigen/ serum ELISA kit (IDEXX HerdCheck Co.) to determine the antigen content of 20 portions of known titter of HCLV semi-cell virus solution which has tested in rabbits, 10 portions of negative HCLV semi-cell virus solution, 10 portions of freeze-dried vaccine and 10 portions of freeze-dried spleen antigen, and compared those results with the results from the thermal reaction in rabbits. As a result, the association rate of the results of semi-finished test and rabbits test was up to 95%, while the association rate of finished test and rabbit test was 75%. ELISA method can be fast, stably and simply used in determination of virus content in semi-finished and finished products of HCLV cell vaccine, and in reverse testing the insensitivity of rabbits test to reduce the testing false and improve quality and yield of the product.