{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T22:35:17Z","timestamp":1792103717900,"version":"4.4.0"},"reference-count":39,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2021,2,7]],"date-time":"2021-02-07T00:00:00Z","timestamp":1612656000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/pathogens10020178","type":"journal-article","created":{"date-parts":[[2021,2,14]],"date-time":"2021-02-14T08:53:56Z","timestamp":1613292836000},"page":"178","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":33,"title":["Characterization of Anti-p54 Monoclonal Antibodies and Their Potential Use for African Swine Fever Virus Diagnosis"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0695-2171","authenticated-orcid":false,"given":"Weldu","family":"Tesfagaber","sequence":"first","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"},{"name":"Department of Veterinary Science, Hamelmalo Agricultural College, Keren 397, Eritrea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lulu","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ghebremedhin","family":"Tsegay","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"},{"name":"Department of Veterinary Science, Hamelmalo Agricultural College, Keren 397, Eritrea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yibrah Tekle","family":"Hagoss","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenjiang","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiwen","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haoyue","family":"Huangfu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fei","family":"Xi","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fang","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Encheng","family":"Sun","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhigao","family":"Bu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7161-6214","authenticated-orcid":false,"given":"Dongming","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,2,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"640","DOI":"10.1126\/science.aaz1439","article-title":"Architecture of African swine fever virus and implications for viral assembly","volume":"366","author":"Wang","year":"2019","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/j.virusres.2012.09.016","article-title":"African swine fever virus morphogenesis","volume":"173","author":"Salas","year":"2013","journal-title":"Virus Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1016\/j.virusres.2012.10.026","article-title":"Pathogenesis of African swine fever in domestic pigs and European wild boar","volume":"173","author":"Blome","year":"2013","journal-title":"Virus Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"282","DOI":"10.3389\/fvets.2020.00282","article-title":"Comparative Pathology and Pathogenesis of African Swine Fever Infection in Swine","volume":"7","author":"Salguero","year":"2020","journal-title":"Front. Vet. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"S\u00e1nchez-Cord\u00f3n, P.J., N\u00fa\u00f1ez, A., Neimanis, A.S., Wikstr\u00f6m-Lassa, E., Montoya, M., Crooke, H., and Gavier-Wid\u00e9n, D. (2019). African Swine Fever: Disease Dynamics in Wild Boar Experimentally Infected with ASFV Isolates Belonging to Genotype I and II. Viruses, 11.","DOI":"10.3390\/v11090852"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.tvjl.2017.12.025","article-title":"African swine fever: A re-emerging viral disease threatening the global pig industry","volume":"233","author":"Montoya","year":"2018","journal-title":"Vet. J."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1482","DOI":"10.1111\/tbed.12989","article-title":"Emergence of African Swine Fever in China","volume":"65","author":"Zhou","year":"2018","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1318","DOI":"10.3389\/fimmu.2019.01318","article-title":"Identification and Immunogenicity of African Swine Fever Virus Antigens","volume":"10","author":"Netherton","year":"2019","journal-title":"Front. Immunol."},{"key":"ref_9","unstructured":"OIE (2020, December 15). Global Situation of African Swine Fever. Available online: https:\/\/rr-asia.oie.int\/en\/projects\/asf\/situational-updates-of-asf-in-asia-and-the-pacific\/."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"950","DOI":"10.1128\/JCM.44.3.950-956.2006","article-title":"Antigenic Properties and Diagnostic Potential of African Swine Fever Virus Protein pp62 Expressed in Insect Cells","volume":"44","author":"Gallardo","year":"2006","journal-title":"J. Clin. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1012","DOI":"10.1128\/CVI.00408-08","article-title":"Recombinant Antigen Targets for Serodiagnosis of African Swine Fever","volume":"16","author":"Gallardo","year":"2009","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3114","DOI":"10.1128\/JCM.00406-06","article-title":"Optimization and Validation of Recombinant Serological Tests for African Swine Fever Diagnosis Based on Detection of the p30 Protein Produced in Trichoplusia ni Larvae","volume":"44","author":"Camacho","year":"2006","journal-title":"J. Clin. Microbiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/j.virusres.2012.10.021","article-title":"African swine fever virus serodiagnosis: A general review with a focus on the analyses of African serum samples","volume":"173","author":"Cubillos","year":"2013","journal-title":"Virus Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/S0166-0934(96)02140-4","article-title":"High level expression of the major antigenic African swine fever virus proteins p54 and p30 in baculovirus and their potential use as diagnostic reagents","volume":"64","author":"Oviedo","year":"1997","journal-title":"J. Virol. Methods"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1681","DOI":"10.1007\/s007050170056","article-title":"Antigenic and immunogenic properties of a chimera of two immunodominant African swine fever virus proteins","volume":"146","author":"Barderas","year":"2001","journal-title":"Arch. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"7244","DOI":"10.1128\/jvi.68.11.7244-7252.1994","article-title":"Characterization and molecular basis of heterogeneity of the African swine fever virus envelope protein p54","volume":"68","author":"Rodriguez","year":"1994","journal-title":"J. Virol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/0168-1702(92)90076-L","article-title":"Cell culture propagation modifies the African swine fever virus replication phenotype in macrophages and generates viral subpopulations differing in protein p54","volume":"23","author":"Alcaraz","year":"1992","journal-title":"Virus Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1006\/viro.1998.9068","article-title":"The African Swine Fever Virus Proteins p54 and p30 Are Involved in Two Distinct Steps of Virus Attachment and Both Contribute to the Antibody-Mediated Protective Immune Response","volume":"243","author":"Oviedo","year":"1998","journal-title":"Virology"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/0168-1702(95)01268-0","article-title":"The structural protein p54 is essential for African swine fever virus viability","volume":"40","author":"Rodriguez","year":"1996","journal-title":"Virus Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4299","DOI":"10.1128\/JVI.78.8.4299-4313.2004","article-title":"African Swine Fever Virus Structural Protein p54 Is Essential for the Recruitment of Envelope Precursors to Assembly Sites","volume":"78","author":"Salas","year":"2004","journal-title":"J. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"9819","DOI":"10.1128\/JVI.75.20.9819-9827.2001","article-title":"African Swine Fever Virus Protein p54 Interacts with the Microtubular Motor Complex through Direct Binding to Light-Chain Dynein","volume":"75","author":"Alonso","year":"2001","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.febslet.2010.11.027","article-title":"Structural models of DYNLL1 with interacting partners: African swine fever virus protein p54 and postsynaptic scaffolding protein gephyrin","volume":"585","author":"Bruix","year":"2011","journal-title":"FEBS Lett."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1331","DOI":"10.1099\/0022-1317-83-6-1331","article-title":"Identification of the principal serological immunodeterminants of African swine fever virus by screening a virus cDNA library with antibody","volume":"83","author":"Kollnberger","year":"2002","journal-title":"J. Gen. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2426","DOI":"10.1099\/vir.0.82857-0","article-title":"Systematic analysis of longitudinal serological responses of pigs infected experimentally with African swine fever virus","volume":"88","author":"Reis","year":"2007","journal-title":"J. Gen. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"888","DOI":"10.1128\/CVI.00395-16","article-title":"Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens","volume":"23","author":"Lokhandwala","year":"2016","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1016\/0166-0934(94)00150-F","article-title":"Highly specific confirmatory Western blot test for African swine fever virus antibody detection using the recombinant virus protein p54","volume":"52","author":"Alcaraz","year":"1995","journal-title":"J. Virol. Methods"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"469","DOI":"10.20506\/rst.17.2.1119","article-title":"Validation of serological assays for diagnosis of infectious diseases","volume":"17","author":"Jacobson","year":"1998","journal-title":"Rev. Sci. Tech."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Alejo, A., Matamoros, T., Guerra, M., and Andr\u00e9s, G. (2018). A Proteomic Atlas of the African Swine Fever Virus Particle. J. Virol., 92.","DOI":"10.1128\/JVI.01293-18"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1111\/tbed.13364","article-title":"Current status and evolving approaches to African swine fever vaccine development","volume":"67","author":"Teklue","year":"2020","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_30","unstructured":"OIE (2012). African Swine Fever. OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, World Organization for Animal Health (OIE)."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.jcpa.2014.09.003","article-title":"An Update on the Epidemiology and Pathology of African Swine Fever","volume":"152","author":"Mur","year":"2015","journal-title":"J. Comp. Pathol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1665","DOI":"10.1111\/tbed.12717","article-title":"Diagnostic specificity of the African swine fever virus antibody detection enzyme-linked immunosorbent assay in feral and domestic pigs in the United States","volume":"64","author":"Bergeron","year":"2017","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"623","DOI":"10.1007\/s11427-020-1657-9","article-title":"A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs","volume":"63","author":"Chen","year":"2020","journal-title":"Sci. China Life Sci."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1080\/22221751.2019.1590128","article-title":"Replication and virulence in pigs of the first African swine fever virus isolated in China","volume":"8","author":"Zhao","year":"2019","journal-title":"Emerg. Microbes Infect."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1080\/22221751.2019.1565915","article-title":"Genome sequences derived from pig and dried blood pig feed samples provide important insights into the transmission of Africa swine fever virus in China in 2018","volume":"8","author":"Wen","year":"2019","journal-title":"Emerg. Microbes Infect."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2172","DOI":"10.1073\/pnas.86.7.2172","article-title":"One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution","volume":"86","author":"Chung","year":"1989","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/0022-1759(80)90146-5","article-title":"Production of monoclonal antibodies: Strategy and tactics","volume":"35","author":"Groth","year":"1980","journal-title":"J. Immunol. Methods"},{"key":"ref_38","first-page":"51","article-title":"Methods in Molecular Biology","volume":"Volume 1318","author":"Hnasko","year":"2015","journal-title":"ELISA"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Wang, L., Mi, S., Gong, W., Shi, J., Madera, R., Ganges, L., Borca, M.V., Ren, J., Cunningham, C., and Cino-Ozuna, A.G. (2020). A neutralizing monoclonal antibody-based competitive ELISA for classical swine fever C-strain post-vaccination monitoring. BMC Vet. Res., 16.","DOI":"10.1186\/s12917-020-2237-6"}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/10\/2\/178\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:20:54Z","timestamp":1760160054000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/10\/2\/178"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,2,7]]},"references-count":39,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2021,2]]}},"alternative-id":["pathogens10020178"],"URL":"https:\/\/doi.org\/10.3390\/pathogens10020178","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,2,7]]}}}