Preparation of Monoclonal Antibodies against the Viral p54 Protein and a Blocking ELISA for Detection of the Antibody against African Swine Fever Virus

Abstract

African swine fever virus (ASFV) causes a highly contagious viral disease in domestic and wild pigs, leading to serious economic losses. As there are no vaccines or drugs available, early accurate diagnosis and eradiation of infected animals are the most important measures for ASFV prevention and control. Therefore, improvement of available diagnostic assays and development of novel effective techniques are required. This study is devoted to generating a new detection platform of blocking monoclonal antibody−based enzyme−linked immunosorbent assay (ELISA) against ASFV p54 protein. Seven monoclonal antibodies against recombinant p54 protein were produced and four epitopes were identified. Three blocking ELISAs were developed with 6A5 and 6F9 mAbs labeled with HRP, respectively, of which the 6A5/6F9−based blocking ELISA displayed the best detection performance, with an AUC of 0.986, sensitivity of 98.36% and specificity of 92.36% in ROC analysis. Moreover, it has an excellent agreement at 96.59% (198/205) when compared to the commercial blocking ELISA (kappa value = 0.920). The method also has high repeatability, with CV <10%, and no cross reaction with the serum antibodies against PRV, PRRSV, CSFV, PCV2 or SVA. This indicates that the 6A5/6F9−based blocking ELISA has high accuracy with good sensitivity and specificity, suitable for viral detection, field surveillance and epidemiological studies.

Preparation and preliminary application of a monoclonal antibody against the African swine fever virus D205R protein

“…However, few effective vaccines or drugs to prevent or control ASF have been developed. Many proteins have been developed as serological diagnostic targets, including P72, P54, and CD2v [20][21][22]. However, due to the large genome of ASFV and complexity of the encoded proteins, more diagnostic targets need to be explored.…”

A novel conserved B-cell epitope in pB602L of African swine fever virus

“…p30 is abundantly expressed in early cell stages, has good antigenicity, and is also an important target for early virus diagnosis (Wang et al 2022 ). Anti-p54 antibodies appear as early as 10 days after ASFV infection, but p54 is prone to false negative results due to amino acid sequence variations, and is not typically used as an ASF detection antigen (Alonso et al 2001 ; Gao et al 2022 ). The CD2v protein, a host immunoregulatory protein, is a serotype-specific cross-protective antigen of ASF and an important protective antigen against ASF (Gladue et al 2020 ; Petrovan et al 2022 ; Sanna et al 2017 ; Song et al 2023 ).…”

Novel Epitope Mapping of African Swine Fever Virus pI215L Protein Using Monoclonal Antibodies

“…Serological assays used for antibody detection are widely applied during ASFV surveillance, ASFV infection induces quick and enduring antibody response [ 22 ]. MAbs against ASFV major capsid protein p72, the structural and highly immunogenic protein p30, and the type II transmembrane protein p54 which plays a key role in virus morphogenesis and viral infection have mostly been used as targets for the detection of ASFV infection in serological assays [ 10 , 23 , 24 , 25 ]. ASFV pI215L protein was abundant in virus-infected PAMs [ 14 ] and exhibited good antigenicity as shown in Figure 1 C. The potential of pI215L protein as a target for early detection of ASFV infection and its sensitivity compared to p30 and p72 is worth exploring.…”

“…ASFV is a large, enveloped virus with icosahedral morphology and belongs to the Asfarviridae family, isolates of which have linear dsDNA genomes of 170–194 kbp, encoding more than 150 polypeptides [ 5 ]. Monoclonal antibodies against various ASFV proteins, including the major capsid protein p72, the viral internal envelope protein p30 and p17, the outer envelope protein CD2v, the type II transmembrane protein p54, the nonstructural protein pK205R, and the uncharacterized protein pCP312R and pC129R, have been generated for ASFV infection research and development of ASFV diagnosis [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. pI215L, a nonstructural protein of ASFV, is found within the virion and expressed at a high level in infected porcine alveolar macrophages (PAMs) from 4 h post-infection (hpi) [ 14 , 15 ], indicating a possible role of pI215L protein in the early steps of infection.…”