Study Of ELISA Antibody Detection Method And Monoclonal Antibody Based On African Swine Fever Virus A137R Antigen

African swine fever(ASF)is a highly contagious pig disease caused by the African swine fever virus(ASFV),which is the number one infectious disease that poses a threat to the global pig industry.The World Organization for Animal Health(WOAH)classifies ASF as a notifiable animal disease,and it is also classified as a class I animal disease in China.In 2018,the first case in China was discovered in Shenyang,and it quickly spread to provinces,cities,and autonomous regions.Currently,the ASF epidemic is generally under control.During the early stages of ASF outbreak,pigs exhibit a short course of disease with high mortality rates and acute symptoms.Often,pigs die without producing antibodies,and rapid nucleic acid detection techniques such as fluorescent PCR are primarily used for pathogen diagnosis.In 2020,a low virulence ASFV genotype II variant strain and genotype I strain were found in China.The ASF epidemic in China has undergone a significant transformation,with the emergence of chronic and asymptomatic infections that are more covertly transmitted and difficult to control.Asymptomatic infected pigs do not have obvious viremia and exhibit irregular shedding of the virus.Relying solely on pathogen diagnosis may lead to missed cases.As infection time prolongs,the body may produce antibodies against the virus,and convenient serological methods can be used for ASF diagnosis.In this study,we conducted reactivity tests using laboratory-prepared ASFV-positive serum and various expressed antigens,virus-infected cells and tissues collected.Through IFA,WB and IHC detection,we found that ASFV-positive serum,serum from pigs infected with a CD2 v gene-deleted strain,and serum from piglets produced after artificial infection with a CD2 v gene-deleted strain all had good specificity reaction with ASFV-infected cells and virus in pig tissues.In WB detection of various expressed ASFV proteins,we found that an early protein p30 and a late protein A137 R had good serum reactivity.The results of the study indicate that laboratory-prepared ASFV-positive serum can be used as a reference serum for the research and evaluation of methods for detecting antibodies in ASFV.A137R is a late protein associated with the virulence of ASFV.This study found that the A137 R protein expressed in E.coli had good reactivity with ASFV recovery serum and could serve as a target antigen for serological detection methods.Monoclonal antibodies prepared using this antigen can be used as experimental materials for studying the pathogenesis of ASFV.An indirect ELISA detection method was established using the ASFVA137 R protein as the raw material,and the optimal coating protein concentration,blocking solution conditions,pig serum dilution concentration,and enzyme-labeled secondary antibody dilution were optimized.The specificity,sensitivity,and compliance of the established method were verified,and it was found that the method could detect positive serum with a maximum dilution of 1:12800 and had no cross-reaction with positive pig sera for other diseases.The agreement rate with commercial reagents was 98.39%.Using the established method to test animal infection serum at different times,it was found that the A137 R antibody reached detectable levels at 13 days post-infection in pigs infected with ASFV.Meanwhile,mice were immunized with A137 R protein as an antigen,and two monoclonal antibodies,1E2 and 1G7,were successfully screened using cell fusion and subcloning,both of which belonged to the Ig G1 subtype.Validation of the obtained monoclonal antibodies showed that they both reacted with virus-infected cell cultures and pig spleens infected with the virus.This study standardized the ASFV-positive serum by infecting cells with the virus,preparing virus-infected pig tissue sections,analyzing SDS electrophoresis bands of virus cell cultures,and conducting reactive tests for ASF antigens.This provides a positive reference serum for ASF serological diagnostic research and evaluation.The study found that the expression of A137 R antigen in Escherichia coli had good reactivity and based on this protein,an indirect ELISA detection method was successfully established.The specificity,sensitivity,and repeatability of the method were studied,which enriched the ASF serological detection methods.Two monoclonal antibodies were successfully prepared using A137 R as the antigen and their characteristics were identified,providing fundamental experimental materials for ASFV basic research.