Establishment and characterization of an immortalized red river hog blood-derived macrophage cell line.

Red river hogs (RRHs) (Potamochoerus porcus), a wild species of Suidae living in Africa with a major distribution in the Guinean and Congolian forests, are natural reservoirs of African swine fever virus (ASFV) and typically are asymptomatic. Since blood and tissue macrophages of suid animals are target cell lineages of ASFV, RRH-derived macrophages are expected to play an important role in suppressing disease development in infected individuals. In the present study, we successfully isolated RRH-derived blood macrophages using co-culture techniques of RRH blood cells with porcine kidney-derived feeder cells and immortalized them by transferring SV40 large T antigen and porcine telomerase reverse transcriptase genes. The newly established macrophage cell line of the RRH-derived blood cell origin (RZJ/IBM) exhibited an Iba1-, CD172a-, and CD203a-positive typical macrophage-like phenotype and up-regulated the phosphorylation of nuclear factor-κB p65 subunit and p38 mitogen-activated protein kinase in response to the bacterial cell wall components, lipopolysaccharide (LPS) and muramyl dipeptide. In addition, RZJ/IBM cells produced the precursor form of interleukin (IL)-1ß and IL-18 upon a stimulation with LPS, leading to the conversion of IL-18, but not IL-1ß, into the mature form. Time-lapse live cell imaging with pHrodo dye-conjugated Escherichia coli BioParticles demonstrated the phagocytotic activity of RZJ/IBM cells. It is important to note that RZJ/IBM cells are clearly susceptible to ASFV infection and support viral replication in vitro. Therefore, the RZJ/IBM cell line provides a unique model for investigating the pathogenesis of ASFV.

Development of quantum dot-based immunochromatographic strip for detection of antibodies against ASFV pp62.

ASFV is the only known double-stranded insect-borne DNA virus, which can rapidly infect domestic pigs and wild boars with ticks as transmission medium. Since it was first discovered in 1921, it quickly spread to all parts of the world and brought huge economic losses to the pig industry all over the world. At present, there is still no safe and effective vaccine for ASFV. Here, we developed a quantum-dot labeled antibody test strip for the detection of antibodies against ASFV pp62. The pp62 protein was labeled with quantum dots, and the antibody test strip was developed uses it in a detection mode of labeled antigen-SPA interceptor-monoclonal antibody quality control. The test strip showed high sensitivity, the positive detection limit of the strip was 1: 106 by continuous multiple dilution using the positive standard serum of ASFV antibody as reference. The test strip showed good specificity, and there was no cross reaction with other swine diseases virus (PCV2, PRRSV, CSFV, PPV). Using the detection results of commercialized kit for African swine fever virus as reference, 80 ASFV antibody negative serum and 4 different ASFV antibody positive serum were detected using the ASFV pp62 quantum-dot labeled antibody test strip. The results were consistent with the commercial kit. This study provides a new detection method for the prevention and control of African swine fever.

Development of plate-type and tubular chemiluminescence immunoassay against African swine fever virus p72.

African swine fever (ASF) is a highly contagious and fatal viral disease that has caused huge economic losses to the pig and related industries worldwide. At present, rapid, accurate, and sensitive laboratory detection technologies are important means of preventing and controlling ASF. However, because attenuated strains of African swine fever virus (ASFV) are constantly emerging, an ASFV antibody could be used more effectively to investigate the virus and control the disease on pig farms. The isolation of ASFV-specific antibodies is also essential for the diagnosis of ASF. Therefore, in this study, we developed two chemiluminescence immunoassays (CLIAs) to detect antibodies directed against ASFV p72: a traditional plate-type blocking CLIA (p72-CLIA) and an automatic tubular competitive CLIA based on magnetic particles (p72-MPCLIA). We compared the diagnostic performance of these two methods to provide a feasible new method for the effective prevention and control of ASF and the purification of ASFV. The cut-off value, diagnostic sensitivity (Dsn), and diagnostic specificity (Dsp) of p72-CLIA were 40%, 100%, and 99.6%, respectively, in known background serum, whereas those of p72-MPCLIA were 36%, 100%, and 99.6%, respectively. Thus, both methods show good Dsn, Dsp, and repeatability. However, when analytical sensitivity was evaluated, p72-MPCLIA was more sensitive than p72-CLIA or a commercial enzyme-linked immunosorbent assay. More importantly, p72-MPCLIA reduced the detection time to 15 min and allowed fully automated detection. In summary, p72-MPCLIA showed superior diagnostic performance and offered a new tool for detecting ASFV infections in the future. KEY POINTS: • Two chemiluminescence immunoassay (plate-type CLIA and tubular CLIA) methods based on p72 monoclonal antibody (mAb) were developed to detect ASFV antibody. • Both methods show good diagnostic performance (Dsn (100%), Dsp (99.6%), and good repeatability), and p72-MPCLIA detects antibodies against ASFV p72 with high efficiency in just 15 min.

Specific Monoclonal Antibodies against African Swine Fever Virus Protease pS273R Revealed a Novel and Conserved Antigenic Epitope.

The African swine fever virus (ASFV) is a large enveloped DNA virus that causes a highly pathogenic hemorrhagic disease in both domestic pigs and wild boars. The ASFV genome contains a double-stranded DNA encoding more than 150 proteins. The ASFV possesses only one protease, pS273R, which is important for virion assembly and host immune evasion. Therefore, the specific monoclonal antibody (mAb) against pS273R is useful for ASFV research. Here, we generated two specific anti-pS273R mAbs named 2F3 and 3C2, both of which were successfully applied for ELISA, Western blotting, and immunofluorescence assays. Further, we showed that both 2F3 and 3C2 mAbs recognize a new epitope of N terminal 1-25 amino acids of pS273R protein, which is highly conserved across different ASFV strains including all genotype I and II strains. Based on the recognized epitope, an indirect ELISA was established and was effective in detecting antibodies during ASFV infection. To conclude, the specific pS273R mAbs and corresponding epitope identified will strongly promote ASFV serological diagnosis and vaccine research.

Defining correlates of protection for mammalian livestock vaccines against high-priority viral diseases.

Enhancing livestock biosecurity is critical to safeguard the livelihoods of farmers, global and local economies, and food security. Vaccination is fundamental to the control and prevention of exotic and endemic high-priority infectious livestock diseases. Successful implementation of vaccination in a biosecurity plan is underpinned by a strong understanding of correlates of protection-those elements of the immune response that can reliably predict the level of protection from viral challenge. While correlates of protection have been successfully characterized for many human viral vaccines, for many high-priority livestock viral diseases, including African swine fever and foot and mouth disease, they remain largely uncharacterized. Current literature provides insights into potential correlates of protection that should be assessed during vaccine development for these high-priority mammalian livestock viral diseases. Establishment of correlates of protection for biosecurity purposes enables immune surveillance, rationale for vaccine development, and successful implementation of livestock vaccines as part of a biosecurity strategy.

Identification and Characterization of a Novel B Cell Epitope of ASFV Virulence Protein B125R Monoclonal Antibody.

The African swine fever virus (ASFV) is an ancient, structurally complex, double-stranded DNA virus that causes African swine fever. Since its discovery in Kenya and Africa in 1921, no effective vaccine or antiviral strategy has been developed. Therefore, the selection of more suitable vaccines or antiviral targets is the top priority to solve the African swine fever virus problem. B125R, one of the virulence genes of ASFV, encodes a non-structural protein (pB125R), which is important in ASFV infection. However, the epitope of pB125R is not well characterized at present. We observed that pB125R is specifically recognized by inactivated ASFV-positive sera, suggesting that it has the potential to act as a protective antigen against ASFV infection. Elucidation of the antigenic epitope within pB125R could facilitate the development of an epitope-based vaccine targeting ASFV. In this study, two strains of monoclonal antibodies (mAbs) against pB125R were produced by using the B cell hybridoma technique, named 9G11 and 15A9. The antigenic epitope recognized by mAb 9G11 was precisely located by using a series of truncated ASFV pB125R. The 52DPLASQRDIYY62 (epitope on ASFV pB125R) was the smallest epitope recognized by mAb 9G11 and this epitope was highly conserved among different strains. The key amino acid sites were identified as D52, Q57, R58, and Y62 by the single-point mutation of 11 amino acids of the epitope by alanine scanning. In addition, the immunological effects of the epitope (pB125R-DY) against 9G11 were evaluated in mice, and the results showed that both full-length pB125R and the epitope pB125R-DY could induce effective humoral and cellular immune responses in mice. The mAbs obtained in this study reacted with the eukaryotic-expressed antigen proteins and the PAM cell samples infected with ASFV, indicating that the mAb can be used as a good tool for the detection of ASFV antigen infection. The B cell epitopes identified in this study provide a fundamental basis for the research and development of epitope-based vaccines against ASFV.

A Non-Hemadsorbing Live-Attenuated Virus Vaccine Candidate Protects Pigs against the Contemporary Pandemic Genotype II African Swine Fever Virus.

African swine fever (ASF) is a highly contagious and severe hemorrhagic transboundary swine viral disease with up to a 100% mortality rate, which leads to a tremendous socio-economic loss worldwide. The lack of safe and efficacious ASF vaccines is the greatest challenge in the prevention and control of ASF. In this study, we generated a safe and effective live-attenuated virus (LAV) vaccine candidate VNUA-ASFV-LAVL3 by serially passaging a virulent genotype II strain (VNUA-ASFV-L2) in an immortalized porcine alveolar macrophage cell line (3D4/21, 50 passages). VNUA-ASFV-LAVL3 lost its hemadsorption ability but maintained comparable growth kinetics in 3D4/21 cells to that of the parental strain. Notably, it exhibited significant attenuation of virulence in pigs across different doses (103, 104, and 105 TCID50). All vaccinated pigs remained healthy with no clinical signs of African swine fever virus (ASFV) infection throughout the 28-day observation period of immunization. VNUA-ASFV-LAVL3 was efficiently cleared from the blood at 14-17 days post-infection, even at the highest dose (105 TCID50). Importantly, the attenuation observed in vivo did not compromise the ability of VNUA-ASFV-LAVL3 to induce protective immunity. Vaccination with VNUA-ASFV-LAVL3 elicited robust humoral and cellular immune responses in pigs, achieving 100% protection against a lethal wild-type ASFV (genotype II) challenge at all tested doses (103, 104, and 105 TCID50). Furthermore, a single vaccination (104 TCID50) provided protection for up to 2 months. These findings suggest that VNUA-ASFV-LAVL3 can be utilized as a promising safe and efficacious LAV candidate against the contemporary pandemic genotype II ASFV.

The antibodies against the A137R protein drive antibody-dependent enhancement of African swine fever virus infection in porcine alveolar macrophages.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a highly contagious disease that can kill up to 100% of domestic pigs and wild boars. It has been shown that the pigs inoculated with some ASF vaccine candidates display more severe clinical signs and die earlier than do pigs not immunized. We hypothesize that antibody-dependent enhancement (ADE) of ASFV infection may be caused by the presence of some unidentified antibodies. In this study, we found that the ASFV-encoded structural protein A137R (pA137R) can be recognized by the anti-ASFV positive sera, indicating that the anti-pA137R antibodies are induced in the ASFV-infected pigs. Interestingly, our results demonstrated that the anti-pA137R antibodies produced in rabbits or pigs enhanced viral replication of different ASFV strains in primary porcine alveolar macrophages (PAMs), the target cells of ASFV. Mechanistic investigations revealed that anti-pA137R antibodies were able to promote the attachment of ASFV to PAMs and two types of Fc gamma receptors (FcγRs), FcγRII and FcγRIII, mediated the ADE of ASFV infection. Taken together, anti-pA137R antibodies are able to drive ASFV ADE in PAMs. These findings shed new light on the roles of anti-ASFV antibodies and have implications for the pathophysiology of the disease and the development of ASF vaccines.

Pathogenicity and virulence of African swine fever virus.

African swine fever (ASF) is a devastating disease with a high impact on the pork industry worldwide. ASF virus (ASFV) is a very complex pathogen, the sole member of the family Asfaviridae, which induces a state of immune suppression in the host through infection of myeloid cells and apoptosis of lymphocytes. Moreover, haemorrhages are the other main pathogenic effect of ASFV infection in pigs, related to the infection of endothelial cells, as well as the activation and structural changes of this cell population by proinflammatory cytokine upregulation within bystander monocytes and macrophages. There are still many gaps in the knowledge of the role of proteins produced by the ASFV, which is related to the difficulty in producing a safe and effective vaccine to combat the disease, although few candidates have been approved for use in Southeast Asia in the past couple of years.

Six adenoviral vectored African swine fever virus genes protect against fatal disease caused by genotype I challenge.

African swine fever virus causes a lethal hemorrhagic disease in domestic swine and wild boar for which currently licensed commercial vaccines are only available in Vietnam. Development of subunit vaccines is complicated by the lack of information on protective antigens as well as suitable delivery systems. Our previous work showed that a pool of eight African swine fever virus genes vectored using an adenovirus prime and modified vaccinia virus boost could prevent fatal disease after challenge with a virulent genotype I isolate of the virus. Here, we identify antigens within this pool of eight that are essential for the observed protection and demonstrate that adenovirus-prime followed by adenovirus-boost can also induce protective immune responses against genotype I African swine fever virus. Immunization with a pool of adenoviruses expressing individual African swine fever virus genes partially tailored to genotype II virus did not protect against challenge with genotype II Georgia 2007/1 strain, suggesting that different antigens may be required to induce cross-protection for genetically distinct viruses. IMPORTANCE: African swine fever virus causes a lethal hemorrhagic disease in domestic pigs and has killed millions of animals across Europe and Asia since 2007. Development of safe and effective subunit vaccines against African swine fever has been problematic due to the complexity of the virus and a poor understanding of protective immunity. In a previous study, we demonstrated that a complex combination of eight different virus genes delivered using two different viral vector vaccine platforms protected domestic pigs from fatal disease. In this study, we show that three of the eight genes are required for protection and that one viral vector is sufficient, significantly reducing the complexity of the vaccine. Unfortunately, this combination did not protect against the current outbreak strain of African swine fever virus, suggesting that more work to identify immunogenic and protective viral proteins is required to develop a truly effective African swine fever vaccine.

Thoughts on the research of African swine fever live-attenuated vaccines.

African swine fever (ASF) is a contagious and fatal disease caused by the African swine fever virus (ASFV), which can infect pigs of all breeds and ages. Most infected pigs have poor prognosis, leading to substantial economic losses for the global pig industry. Therefore, it is imperative to develop a safe and efficient commercial vaccine against ASF. The development of ASF vaccine can be traced back to 1960. However, because of its large genome, numerous encoded proteins, and complex virus particle structure, currently, no effective commercial vaccine is available. Several strategies have been applied in vaccine design, some of which are potential candidates for vaccine development. This review provides a comprehensive analysis on the safety and effectiveness, suboptimal immunization effects at high doses, absence of standardized evaluation criteria, notable variations among strains of the same genotype, and the substantial impact of animal health on the protective efficacy against viral challenge. All the information will be helpful to the ASF vaccine development.

Development of novel monoclonal antibodies for blocking NF-κB activation induced by CD2v protein in African swine fever virus.

Background: CD2v, a critical outer envelope glycoprotein of the African swine fever virus (ASFV), plays a central role in the hemadsorption phenomenon during ASFV infection and is recognized as an essential immunoprotective protein. Monoclonal antibodies (mAbs) targeting CD2v have demonstrated promise in both diagnosing and combating African swine fever (ASF). The objective of this study was to develop specific monoclonal antibodies against CD2v. Methods: In this investigation, Recombinant CD2v was expressed in eukaryotic cells, and murine mAbs were generated through meticulous screening and hybridoma cloning. Various techniques, including indirect enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence assay (IFA), and bio-layer interferometry (BLI), were employed to characterize the mAbs. Epitope mapping was conducted using truncation mutants and epitope peptide mapping. Results: An optimal antibody pair for a highly sensitive sandwich ELISA was identified, and the antigenic structures recognized by the mAbs were elucidated. Two linear epitopes highly conserved in ASFV genotype II strains, particularly in Chinese endemic strains, were identified, along with a unique glycosylated epitope. Three mAbs, 2B25, 3G25, and 8G1, effectively blocked CD2v-induced NF-κB activation. Conclusions: This study provides valuable insights into the antigenic structure of ASFV CD2v. The mAbs obtained in this study hold great potential for use in the development of ASF diagnostic strategies, and the identified epitopes may contribute to vaccine development against ASFV.

Identification of two novel B cell epitopes on E184L protein of African swine fever virus using monoclonal antibodies.

African swine fever virus (ASFV) is a large double-stranded DNA virus with a complex structural architecture and encodes more than 150 proteins, where many are with unknown functions. E184L has been reported as one of the immunogenic ASFV proteins that may contribute to ASFV pathogenesis and immune evasion. However, the antigenic epitopes of E184L are not yet characterized. In this study, recombinant E184L protein was expressed in prokaryotic expression system and four monoclonal antibodies (mAbs), designated as 1A10, 2D2, 3H6, and 4C10 were generated. All four mAbs reacted specifically with ASFV infected cells. To identify the epitopes of the mAbs, a series of overlapped peptides of E184L were designed and expressed as maltose binding fusion proteins. Accordingly, the expressed fusion proteins were probed with each E184L mAb separately by using Western blot. Following a fine mapping, the minimal linear epitope recognized by mAb 1A10 was identified as 119IQRQGFL125, and mAbs 2D2, 3H6, and 4C10 recognized a region located between 153DPTEFF158. Alignment of amino acids of E184L revealed that the two linear epitopes are highly conserved among different ASFV isolates. Furthermore, the potential application of the two epitopes in ASFV diagnosis was assessed through epitope-based ELISA using 24 ASFV positive and 18 negative pig serum and the method were able to distinguish positive and negative samples, indicating the two epitopes are dominant antigenic sites. To our knowledge, this is the first study to characterize the B cell epitopes of the antigenic E184L protein of ASFV, offering valuable tools for future research, as well as laying a foundation for serological diagnosis and epitope-based marker vaccine development.

Characterization of an African swine fever virus outbreak in India and comparative analysis of immune genes in infected and surviving crossbreed vs. indigenous Doom pigs.

Since 2020, African swine fever (ASF) has affected all pig breeds in Northeast India except Doom pigs, a unique indigenous breed from Assam and the closest relatives of Indian wild pigs. ASF outbreaks result in significant economic losses for pig farmers in the region. Based on sequencing and phylogenetic analysis of the B646L (p72) gene, it has been determined that ASFV genotype II is responsible for outbreaks in this region. Recent studies have shown that MYD88, LDHB, and IFIT1, which are important genes of the immune system, are involved in the pathogenesis of ASFV. The differential expression patterns of these genes in surviving ASFV-infected and healthy Doom breed pigs were compared to healthy controls at different stages of infection. The ability of Doom pigs to withstand common pig diseases, along with their genetic resemblance to wild pigs, make them ideal candidates for studying tolerance to ASFV infection. In the present study, we investigated the natural resistance to ASF in Doom pigs from an endemic area in Northeast India. The results of this study provide important molecular insights into the regulation of ASFV tolerance genes.

African swine fever virus pB475L evades host antiviral innate immunity via targeting STAT2 to inhibit IFN-I signaling.

African swine fever virus (ASFV) causes severe disease in domestic pigs and wild boars, seriously threatening the development of the global pig industry. Type I interferon (IFN-I) is an important component of innate immunity, inducing the transcription and expression of antiviral cytokines by activating Janus-activated kinase-signal transducer and activator of transcription (STAT). However, the underlying molecular mechanisms by which ASFV antagonizes IFN-I signaling have not been fully elucidated. Therefore, using coimmunoprecipitation, confocal microscopy, and dual luciferase reporter assay methods, we investigated these mechanisms and identified a novel ASFV immunosuppressive protein, pB475L, which interacts with the C-terminal domain of STAT2. Consequently, pB475L inhibited IFN-I signaling by inhibiting STAT1 and STAT2 heterodimerization and nuclear translocation. Furthermore, we constructed an ASFV-B475L7PM mutant strain by homologous recombination, finding that ASFV-B475L7PM attenuated the inhibitory effects on IFN-I signaling compared to ASFV-WT. In summary, this study reveals a new mechanism by which ASFV impairs host innate immunity.

Development of a P30 protein-based indirect ELISA for detecting African swine fever antibodies utilizing the HEK293F expression system.

African swine fever (ASF) is an acute, febrile, and highly lethal infectious disease in pigs caused by the African swine fever virus (ASFV). Effective detection methods and strict biosecurity measures are crucial for preventing and controlling ASF, especially since there are currently no commercially available vaccines or antiviral drugs to combat ASFV infection effectively. However, the emergence of low-virulence strains of ASFV in recent years has led to false-positive results, highlighting the importance of early-produced antibody detection methods. Therefore, detecting antibodies against ASFV produced early in the infection can facilitate the prompt identification of infected pigs. This study focused on the p30 protein, an early expressed protein during ASFV infection, to develop an indirect ELISA. This method was established using the HEK293F suspension cell expression system, which has the ability to produce large quantities of correctly folded proteins with normal functionality. In this study, we developed an indirect ELISA test utilizing the p30 recombinant protein produced by the HEK293F suspension cell expression system as the antigen coating. The concentration of the p30 protein obtained from the HEK293F suspension cell expression system was measured at 4.668 mg/mL, serving as the foundation for establishing the indirect ELISA. Our findings indicate that the indirect ELISA method exhibits a sensitivity of 1:12800. Furthermore, it demonstrates high specificity and excellent reproducibility. Comparing our results to those obtained from the commercial kit, we found a coincidence rate of 98.148 % for the indirect ELISA. In summary, we have developed a sensitive method for detecting ASFV, providing a valuable tool for monitoring ASFV infection in pig herds.

Monoclonal antibody development for early detection of ASFV I73R protein: Identification of a linear antigenic epitope.

African swine fever virus (ASFV), which was first identified in northern China in 2018, causes high mortality in pigs. Since the I73R protein in ASFV is abundantly expressed during the early phase of virus replication, it can be used as a target protein for early diagnosis. In this study, the I73R protein of ASFV was expressed, and we successfully prepared a novel monoclonal antibody (mAb), 8G11D7, that recognizes this protein. Through both indirect immunofluorescence and Western blotting assays, we demonstrated that 8G11D7 can detect ASFV strains. By evaluating the binding of the antibody to a series of I73R-truncated peptides, the definitive epitope recognized by the monoclonal antibody 8G11D7 was determined to be 58 DKTNTIYPP 66. Bioinformatic analysis revealed that the antigenic epitope had a high antigenic index and conservatism. This study contributes to a deeper understanding of ASFV protein structure and function, helping establish ASFV-specific detection method.

African swine fever virus MGF505-6R attenuates type I interferon production by targeting STING for degradation.

African swine fever (ASF) is an acute hemorrhagic and devastating infectious disease affecting domestic pigs and wild boars. It is caused by the African swine fever virus (ASFV), which is characterized by genetic diversity and sophisticated immune evasion strategies. To facilitate infection, ASFV encodes multiple proteins to antagonize host innate immune responses, thereby contributing to viral virulence and pathogenicity. The molecular mechanisms employed by ASFV-encoded proteins to modulate host antiviral responses have not been comprehensively elucidated. In this study, it was observed that the ASFV MGF505-6R protein, a member of the multigene family 505 (MGF505), effectively suppressed the activation of the interferon-beta (IFN-ß) promoter, leading to reduced mRNA levels of antiviral genes. Additional evidence has revealed that MGF505-6R antagonizes the cGAS-STING signaling pathway by interacting with the stimulator of interferon genes (STING) for degradation in the autophagy-lysosomal pathway. The domain mapping revealed that the N-terminal region (1-260aa) of MGF505-6R is the primary domain responsible for interacting with STING, while the CTT domain of STING is crucial for its interaction with MGF505-6R. Furthermore, MGF505-6R also inhibits the activation of STING by reducing the K63-linked polyubiquitination of STING, leading to the disruption of STING oligomerization and TANK binding kinase 1 (TBK1) recruitment, thereby impairing the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3). Collectively, our study elucidates a novel strategy developed by ASFV MGF505-6R to counteract host innate immune responses. This discovery may offer valuable insights for further exploration of ASFV immune evasion mechanisms and antiviral strategies.

Epitope mapping and establishment of a blocking ELISA for mAb targeting the p72 protein of African swine fever virus.

The African swine fever virus (ASFV) has the ability to infect pigs and cause a highly contagious acute fever that can result in a mortality rate as high as 100%. Due to the viral epidemic, the pig industry worldwide has suffered significant financial setbacks. The absence of a proven vaccine for ASFV necessitates the development of a sensitive and reliable serological diagnostic method, enabling laboratories to effectively and expeditiously detect ASFV infection. In this study, four strains of monoclonal antibodies (mAbs) against p72, namely, 5A1, 4C4, 8A9, and 5E10, were generated through recombinant expression of p72, the main capsid protein of ASFV, and immunized mice with it. Epitope localization was performed by truncated overlapping polypeptides. The results indicate that 5A1 and 4C4 recognized the amino acid 20-39 aa, 8A9 and 5E10 are recognized at 263-282 aa, which is consistent with the reported 265-280 aa epitopes. Conserved analysis revealed 20-39 aa is a high conservation of the epitopes in the ASFV genotypes. Moreover, a blocking ELISA assay for detection ASFV antibody based on 4C4 monoclonal antibody was developed and assessed. The receiver-operating characteristic (ROC) was performed to identify the best threshold value using 87 negative and 67 positive samples. The established test exhibited an area under the curve (AUC) of 0.9997, with a 95% confidence interval ranging from 99.87 to 100%. Furthermore, the test achieved a diagnostic sensitivity of 100% (with a 95% confidence interval of 95.72 to 100%) and a specificity of 98.51% (with a 95% confidence interval of 92.02 to 99.92%) when the threshold was set at 41.97%. The inter- and intra-batch coefficient of variation were below 10%, demonstrating the exceptional repeatability of the method. This method can detect the positive standard serum at a dilution as high as 1:512. Subsequently, an exceptional blocking ELISA assay was established with high diagnostic sensitivity and specificity, providing a novel tool for detecting ASFV antibodies. KEY POINTS: • Four strains of ASFV monoclonal antibodies against p72 were prepared and their epitopes were identified. • Blocking ELISA method was established based on monoclonal antibody 4C4 with an identified conservative epitope. • The established blocking ELISA method has a good effect on the detection of ASFV antibody.