Study On The Stability Of Inactivated FMD Antigen And The Alternative Method Of Efficacy Test Of Inactivated Vaccine

【Objective】To study the stability of foot and mouth disease antigen and remove the interference of impurities in the detection process,compare and analyze four detection methods of 146S antigen content of foot and mouth disease,and screen out an easily standardized method for the detection of 146S antigen.The dynamic changes of cytokines in serum of pigs were detected.Compared with the serum antibody assay for immunized animals and the guinea pig immune attack protection test,the alternative method suitable for the animal efficacy test was selected.【Methods】The content of 146S in FMD inactivated antigen was detected by sucrose density gradient centrifugation combined with Agilent Cary 100 ultraviolet spectrophotometer,sucrose density gradient centrifugation combined with Agilent 1260liquid chromatography,high performance liquid chromatography with volume exclusion chromatography and ELISA,and the data of four detection methods were analyzed statistically.The inactivated FMD antigens of four different strains were lysed at 50℃for a certain period of time.One FMD A-antigen and one FMD O-antigen were stored at4℃for 12 months and repetitiously frozen.The FMD antigens were treated with Benzonase nuclease,and then the content of 146S antigen before and after treatment was detected by sucrose density gradient centrifugation combined with Agilent 1260 liquid chromatography.The content of effective antigen 146S in three batches of bivalent inactivated test vaccines of porcine FMD type O and type A was detected by high performance liquid chromatography（HPLC）.The content of type O and type A antigens in the vaccines was detected by ELISA kits for each type of FMD antigen.The dynamic changes of cytokines IFN-γ,IL-4 and IL-10 in serum of immunized pigs were detected by ELISA.The antibody level after 28 days of immunization was detected by liquid-phase blocking ELISA.The efficacy of the vaccine(PD₅₀)was detected by guinea pig immune challenge protection test and the test animal immune challenge protection test respectively.The causal relationships between the antigen content and PD₅₀,between guinea pig immune challenge protection test and the test animal immune challenge protection test,and between antibody levels and PD₅₀ after immunization was analyzed.【Results】The correlations between the values of the four assays were highly significant and positive.FMD antigen strain 1（type Asia）and FMD antigen strain 2（type A）were the most stable and FMD antigen strain 4（type O）was the least stable.The antigen content did not change after being placed at 4°C for 12 months,while the antigen content was more degraded after repetitious freezing treatments.The antigen after Benzonase nuclease treatment contained less impure protein.The antigen content of146S in the three batches of FMD type O and A inactivated test vaccines was 1.08μg,7.48μg and 15.63μg per head,respectively.For the test pig immune challenge test,the half-protective dose(PD₅₀)of the high virulent virus for FMD/O/MYA98/BY/2010CF3test was 7.49,11.84 and 15.59,respectively,and the half-protective dose(PD₅₀)of the high virulent virus for FMD/A/GDMM/2013CF2 test was 7.49,10.81 and 15.59respectively.For the guinea pig immune challenge test,the half-protective dose(PD₅₀)of the high virulent virus for FMD/O/MYA98/BY/2010CF3 test was 9.00,9.00,and 11.84,respectively,and the half-protective dose(PD₅₀)of the high virulent virus for FMD/A/GDMM/2013CF2 test was 7.19,11.84,and 10.32 respectively.Compared to the control group of tested pigs,the levels of IFN-γin the serum of immunized pigs increased significantly,IL-10 levels decreased and IL-4 levels increased significantly.The t-test showed that there was a positive correlation（p<0.01）between the log antibody potency of the test animals and the rate of protection against challenge（probability units）in the test animal,with a significant correlation of the one-dimensional linear regression equation of y=2.436x+1.840 and a correlation coefficient R²=0.462.There was a positive correlation（p<0.01）between guinea pig protection rate against challenge and the protection rate against challenge of the test animal,with a significant correlation of y=1.3491x-1.841 and a correlation coefficient of R²=0.4581.There was a positive correlation（p<0.01）between the 146S content and the protection rate against challenge(PD₅₀)in the three batches of tested vaccines,and the correlation was highly significant,with a one-dimensional linear regression equation of y=0.907x+7.186 and a correlation coefficient R²=0.987.【Conclusion】The method of sucrose density gradient centrifugation combined with Agilent 1260 liquid chromatography is simple and reproducible.The method of HPLC is highly automated,rapid,reproducible and easy to standardize.At 50℃,the stability of FMD-O antigen was the least stable and most susceptible to lysis.FMD antigen or vaccine is suitable for storage at 4℃.Benzonase nuclease can effectively remove the interference of impure protein and improve the efficiency of chromatographic purification.The results showed that the dynamic changes of cytokines in serum of pigs immunized with foot-and-mouth disease vaccine were significant.The dynamic changes of cytokines in porcine serum after FMD vaccine immunization were significant,with the positive correlation between 146S content in three batches of tested vaccines and the protection rate against challenge of the test animals much greater than the correlation between both the protection rate against challenge of guinea pigs and antibody potency with the protection rate against challenge.When the 146S content in each vaccine was more than 1μg,the protection rate could reach more than 6 PD₅₀.The 146S content detection method can be considered as an alternative method to test the efficacy of animal immune challenge test experiment for further research,and the serum antibody detection method is an ideal method to evaluate the immune effect of FMD herd in the field.