Purification Of Fowl Adenovirus And Establishment Of Indirect ELISA Detection Method For Antibodies Against Fowl Adenovirus

Since 2015,chicken hydropericardium and hepatitis syndrome caused by fowl adenovirus?group I,serotype 4?has broken out in many provinces in China.And the incidence rate is on the rise.The disease occurs mainly in 3 to 5 week-old chickens.It is characterized by hydropericardium and inclusion body hepatitis.The diseased chickens show sudden onset and rapidly die.The mortality rate is as high as 30%to 80%.It has caused huge economic losses to the poultry industry.At present,the primary prevention and control measure is vaccination.And the immune effects of vaccines can be evaluated by monitoring antibody levels in vaccinated chickens.Establishing detection methods that can reflect the trend of antibody levels in vaccinated chickens is of great significance for disease prevention and control.Therefore,in this study,the whole virus was used as the coating antigen to establish an indirect ELISA method for detection of antibodies against fowl adenovirus?group I,serotype 4?.And the method was applied in clinical practice.In this study,after purification of fowl adenovirus?group I,serotype 4?by using G200molecular sieve,Macro-prep High Q anion exchange chromatography combined with ultrafiltration,the virus recovery was 31.62%.With the same virus content,the removal rate of protein in the virus solution was 97.56%.The purified fowl adenovirus was used as ELISA coating antigen.Then optimize the coating antigen concentration and coating conditions.The experimental results showed that the coating antigen concentration of 2?g/mL and the coating conditions of 4?overnight were better.To optimize the blocking conditions,the wells were blocked with PBST solution containing 5%FBS overnight at4?.The dilution concentration and reaction conditions of antisera and horseradish peroxidase-labelled rabbit-anti-chicken IgG,and the reaction conditions of TMB substrate were also optimized.The antisera were diluted 2000-fold and incubated for 1 h at 37?.Horseradish peroxidase-labelled rabbit-anti-chicken IgG were diluted 10000-fold and incubated for 1 h at 37?.The reaction conditions of TMB substrate were incubation in the dark for 10 min at room temperature.Under the above conditions,the OD₄₅₀ values of positive sera were greater than 1.5,the OD₄₅₀ values of negative sera were less than 0.2,and the P/N values were large.The specificity and sensitivity of laboratory trial products were studied.The results showed that the ELISA method established in this study did not react with sera positive for AIV,NDV,IBV,IBDV,REV,EDSV?1:2000?or 30 negative sera from SPF chickens?1:2000?.And the OD₄₅₀ values were all less than 0.2.It reacted with antisera against FAdV?1:2000?,and the OD₄₅₀ values were greater than 1.5.While the positive control sera were diluted to 1:12800,the OD₄₅₀ values were still greater than 0.2.According to the dynamic change of antibody levels in vaccinated chickens,which were detected by the optimized ELISA procedures,the cut-off values for the indirect ELISA method established in this study were determined.When the OD₄₅₀ values of positive control sera were greater than1.0 and the OD₄₅₀ values of negative control sera were less than 0.2,the assay was feasible.Serum samples with S/P values greater than or equal to 0.2 were judged to be positive;those with S/P values between 0.1 and 0.2 were judged to be weakly positive;and those with S/P values less than 0.1 were judged to be negative.In addition,the stability of ELISA coated plates was studied.The results showed that the difference between batches was less than 10%,and the difference within batches was less than 5%.The specificity and sensitivity did not change significantly after 12 months of storage.A total of 363 clinical serum samples from Guangdong,Guangxi and Yunnan were detected using the ELISA method established in this study.The detection results were consistent with the dynamic change of antibody levels in vaccinated chickens.All the results showed that the ELISA method established in this study had good sensitivity,specificity,and stability for detection of antibodies against fowl adenovirus.The ELISA method can make up for the shortcomings of commercial kits on the market and has a good market application prospect.