Establishment And Application Of Immunoperoxidase Monolayer Assay For Detecting The Serum Antibody Of African Swine Fever

African swine fever(ASF),caused by African swine fever virus(ASFV),is an acute,severe,highly contagious disease of domestic and wild pigs.The mortality rate of ASF could reach up to 100%,leading to serious economic and production losses,and there is no effective vaccine and medicine till now.As a notorious swine infectious disease worldwide,the ASF epidemic was first reported in Kenya in 1921.ASF,as a foreign animal disease,was first detected in Shenyang,Liaoning Province in China on 3rd August 2018,then spread across the whole country,causing severe economic losses.The natural attenuated strain of type II ASFV was first discovered in China in 2020,most majority of the infected pigs showed mild symptoms,sometimes they can live through with ASF,which made it difficult to be diagnosed.In short,precise ASF prevention and control,as well as the sensitive and specific serological diagnosis method,are urgent to be established.At present,ELISA is the recommended method for serological detection by Office International Des Epizooties(OIE).However,ELISA is not the best choice due to the requirements of precise equipment and complicated preparation of antigens.Compared with ELISA,the immunoperoxidase monolayer assay(IPMA)is a simpler and economical method with better specificity and sensitivity.The results of IPMA always were determined by optical microscope,thus the operation standards are relatively concise.Meanwhile,the results of dyeing in the plate are more stable for further analysis,which provides a more economical and practical method for the detection of large numbers of sample in fields.In 2015,Gallardo demonstrated that ASF-IPT(IPMA)is the most sensitive method among the current serological detection methods.Therefore,the establishment of ASF-IPMA is of great importance for the monitoring,prevention and control of the ASF epidemic.In order to establish ASF-IPMA,two kinds of ASFV attenuated strains etc.the attenuated strain of African swine fever with MGF360-505R gene deletion(r ASFV?360)and the attenuated strain of African swine fever with 7 gene deletion(HLJ/18-7GD),and two kinds of cells etc.PK15 and Vero were compared on biosafety,infection ability cell morphology and chromogenic ability.The results showed that,although HLJ/18-7GD had weaker infection ability than r ASFV?360,the color dyeing ability on the two cells infecting with two viruses respectively were similar in IPMA,but HLJ/18-7GD had weaker virulence and stronger safety.Meanwhile,the morphology and color of PK15 cells was more uniform after dying,which is easier to be observed.Therefore,HLJ/18-7GD and PK15 cell were selected for IPMA.On the basis of the results above,the conditions of ASF-IPMA were further optimized,the infection titer of HLJ/18-7GD virus was 1×10⁷ TCID₅₀/m L,and the virus replication time was 72 h.During the operation of IPMA,the optimal dilution of secondary antibody was 1:2000,and the optimal dilution of serum was 1:100.According to the optimized conditions above,several batches of IPMA reaction plates were prepared and the experiments of specificity,sensitivity and reproducibility of the method were carried out.The results showed that ASF-IPMA had better specificity and had no cross reaction with the sera of other common porcine viruses.The sensitivity of ASF-IPMA was 3.48 times higher than that of P30-i ELISA,and 27.86 times higher than that of P54-i ELISA.And there was no difference between and within batches.Finally,ASF-IPMA was compared with three kits etc.P30-i ELISA,P54-i ELISA and VP72 blocking ELISA,the results shows that the coincidence rate was no less than 98%through testing109 serum samples.In conclusion,this study established an ASF-IPMA detection method,which has good sensitivity,specificity and reproducibility,and is also an economical method.The assembled IPMA kit will provide a new and more effective way for ASF monitoring,prevention and control in China.