Production And Characterization Of Monoclonal Antibodies Against P54/E183L Protein And Their Application For African Swine Fever Virus Diagnosis

African swine fever is a highly lethal hemorrhagic viral disease of domestic pigs caused by African swine fever virus(ASFV)and currently,no effective vaccine is available.Therefore,an early and accurate detection of ASFV is critical for effective disease control program.Several ELISA-based serological tests integrating p72,p30 and pp62 antigens are commercially available.Alternatively,p54/E183 L,a structural and highly immunogenic protein of ASFV,has been also reported as a good candidate for serological diagnosis.Improving the available diagnostic assays and adding some validated techniques are useful for a range of serological investigations.Moreover,studying the antigenic determinants of ASFV proteins and incorporating to the available diagnostic tools will be a step forward for developing a more accurate and reliable diagnostic approach.The aim of the current study was therefore to produce and characterize p54 monoclonal antibodies with an ultimate goal of developing a monoclonal antibody-based ELISA for African swine fever virus antibody detection.Herein,p54/E183 L,a structural protein of ASFV,was successfully constructed,expressed and purified.Likewise,five monoclonal antibodies were generated against Escherichia coli expressed p54 protein and their characterizations were investigated.Western-blot and indirect immunofluorescent assay analysis revealed that the monoclonal antibodies strongly react with the immunizing antigen and ASFV-infected pulmonary alveolar macrophages(PAM cells).Further screening for epitope mapping was performed against three overlapping polypeptides expressed in Escherichia coli.Based on these polypeptide,four anti-p54 monoclonal antibodies(2A7,2D9,3F2 &3D1)were able to recognize only the C-terminal end of p54/E183 L gene(120-184aa).While one monoclonal antibody designated as 4G5 identified the N-terminus part(53-82aa).In addition,the potential use of these five p54 monoclonal antibodies for ASFV antibody detection was investigated and a competitive enzyme-linked immunosorbent assay(c ELISA)based on 2A7 was developed.To evaluate the performance of the assay,a total of 365 pig serum samples(178 negative and 187 positive samples)were tested and a receiver-operating characteristic(ROC)analysis was applied to determine the cut-off value.Based on the ROC analysis,the area under the curve(AUC)was0.982(95% confidence interval: 96.9% to 99.4%),besides a sensitivity of 92.5% and a specificity of98.9% was achieved when the percent inhibition of 20% was selected as a threshold.Moreover,the result showed an excellent agreement when compared to other commercially available blocking ELISA(kappa value = 0.912)and showed no reaction to other swine pathogens.Overall,the newly developed c ELISA method offers a promising approach for a rapid and convenient ASFV serodiagnosis,which could be used as alternative to other serological assays for screening possible ASFV infection.