Characterization of Anti-p54 Monoclonal Antibodies and Their Potential Use for African Swine Fever Virus Diagnosis

Abstract

African swine fever (ASF) is a highly lethal hemorrhagic viral disease of domestic pigs caused by African swine fever virus (ASFV). Although a good advance has been made to understand the virus, a safe and effective vaccine against ASFV is still lacking and its eradication solely depends on its early and accurate diagnosis. Thus, improving the available diagnostic assays and adding some validated techniques are useful for a range of serological investigations. The aim of this study was to produce and characterize p54 monoclonal antibodies with an ultimate goal of developing a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for ASFV antibody detection. Five monoclonal antibodies against p54 protein expressed in Escherichia coli was generated and their characterizations were investigated. Furthermore, a competitive enzyme-linked immunosorbent assay (cELISA) based on a monoclonal antibody designated as 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) was 0.982 (95% confidence interval: 96.9% to 99.4%), besides a sensitivity of 92.5% and a specificity of 98.9% 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 cELISA 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.

Genotype I African swine fever viruses emerged in domestic pigs in China and caused chronic infection

“…PAMs were seeded in 96-well plates and infected with different doses of ASFVs. Virus replication was confirmed by using an Immunofluorescence assay (IFA) with an ASFV-specific antibody, as described previously [ 15 , 18 ]. The 50% tissue culture infective dose (TCID 50 ) was measured by using the Reed and Muench method [ 17 ].…”

Identification of novel linear epitopes in P72 protein of African swine fever virus recognized by monoclonal antibodies

“…Monoclonal antibodies against P72 protein were produced according to the previous method ( Tesfagaber et al, 2021 ). Briefly, the recombinant-P72&B602L expression proteins were emulsified with complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO, USA) at a 1:1 ratio.…”

Development of a potential diagnostic monoclonal antibody against capsid spike protein VP27 of the novel goose astrovirus

“…For example, the Nsp10-specific mAb of porcine reproductive and respiratory syndrome virus ( PRRSV ) may facilitate the establishment of diagnostic methods to discriminate between genotype 1 and genotype 2 PRRSV infection ( Zhang et al, 2017 ). Additionally, Tesfagaber et al developed mAb-based ELISA for African swine fever virus antibody detection ( Tesfagaber et al, 2021 ). mAb-based methods have also been developed for the diagnosis of astrovirus and are based on several techniques, including ELISA, IFA, and IHC ( Herrmann et al, 1988 , 1990 ; Sanchez-Fauquier et al, 1994 ).…”