{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T15:39:07Z","timestamp":1785425947887,"version":"3.56.0"},"reference-count":40,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,6,25]],"date-time":"2026-06-25T00:00:00Z","timestamp":1782345600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"China Agriculture Research System of MOF and MARA","award":["CARS-35"],"award-info":[{"award-number":["CARS-35"]}]},{"award":["CARS-35"],"award-info":[{"award-number":["CARS-35"]}],"id":[{"id":"https:\/\/ror.org\/03p5eky76","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012246","name":"Priority Academic Program Development of Jiangsu Higher Education Institutions","doi-asserted-by":"publisher","award":["PAPD"],"award-info":[{"award-number":["PAPD"]}],"id":[{"id":"10.13039\/501100012246","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012226","name":"Fundamental Research Funds for the Central Universities","doi-asserted-by":"crossref","award":["PY2026009"],"award-info":[{"award-number":["PY2026009"]}],"id":[{"id":"10.13039\/501100012226","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Veterinary Sciences"],"abstract":"<jats:p>Porcine circovirus type 2 (PCV2) is the primary causative agent of a spectrum of porcine circovirus-associated diseases (PCVDs) and remains a major threat to the global swine industry. In this study, ten monoclonal antibodies (mAbs) targeting the Cap protein of PCV2 were generated and characterized. One mAb, designated 4C4, which exhibited high reactivity, strong neutralizing activity, and superior blocking efficacy, was selected for horseradish peroxidase (HRP) labeling. After optimizing the reaction parameters, a blocking ELISA was developed for the detection of the anti-PCV2 antibody. Using receiver operating characteristic (ROC) curve analysis, a cutoff value of 40% was established to distinguish positive from negative serum samples. The sensitivity and specificity of this blocking ELISA method were 98.66% and 100%, respectively. No cross-reactivity was observed with serum antibodies against classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine reproductive and respiratory syndrome virus (PRRSV), or pseudorabies virus (PRV). Intra-assay and inter-assay repeatability tests yielded coefficients of variation (CVs) all below 10%, confirming the assay\u2019s excellent reproducibility. Simultaneous testing of 312 clinical porcine serum samples using the developed bELISA and a commercial indirect ELISA kit revealed an overall coincidence rate of 99.04%. In addition, the percentage inhibition (PI) in the bELISA was strongly correlated with serum anti-PCV2 neutralizing antibody titers. In conclusion, the blocking ELISA developed herein demonstrates high sensitivity, strong specificity, and good reproducibility, serving as a potentially effective tool for the detection of the anti-PCV2 antibody and epidemiological investigation.<\/jats:p>","DOI":"10.3390\/vetsci13070617","type":"journal-article","created":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T00:49:23Z","timestamp":1782434963000},"page":"617","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Preparation of Monoclonal Antibodies Against Porcine Circovirus Type 2 Capsid Protein and Development of a Blocking ELISA for Detection of the Antibody Against the Virus"],"prefix":"10.3390","volume":"13","author":[{"given":"Haifeng","family":"Sun","sequence":"first","affiliation":[{"name":"Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qingqing","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuyan","family":"Zhai","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Biyue","family":"Wu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zicheng","family":"Ma","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yangyang","family":"Sun","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaiyuan","family":"Ye","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haoyuan","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanni","family":"Gao","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xianwei","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"},{"name":"Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Bai","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"},{"name":"Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ping","family":"Jiang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"},{"name":"Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,25]]},"reference":[{"key":"ref_1","first-page":"44","article-title":"Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome","volume":"39","author":"Ellis","year":"1998","journal-title":"Can. Vet. J."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"3443","DOI":"10.1016\/j.vaccine.2008.04.032","article-title":"Reduction of PMWS-associated clinical signs and co-infections by vaccination against PCV2","volume":"26","author":"Ritzmann","year":"2008","journal-title":"Vaccine"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.virusres.2011.10.007","article-title":"Porcine circovirus type 2 (PCV2) infections: Clinical signs, pathology and laboratory diagnosis","volume":"164","year":"2012","journal-title":"Virus Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1016\/j.rvsc.2004.03.007","article-title":"The presence of co-infections in pigs with clinical signs of PMWS in The Netherlands: A case-control study","volume":"77","author":"Wellenberg","year":"2004","journal-title":"Res. Vet. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"5262","DOI":"10.1128\/JVI.72.6.5262-5267.1998","article-title":"Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs","volume":"72","author":"Hamel","year":"1998","journal-title":"J. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Franzo, G., and Segal\u00e9s, J. (2018). Porcine circovirus 2 (PCV-2) genotype update and proposal of a new genotyping methodology. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0208585"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Lamas, A., Garrido-Maestu, A., and L\u00f3pez-Lorenzo, G. (2025). Environmental and Serological Monitoring of Porcine Circovirus by Loop-Mediated Isothermal Amplification in Pig Farms. Vet. Sci., 12.","DOI":"10.3390\/vetsci12101011"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Nainys, J., Lasickiene, R., Petraityte-Burneikiene, R., Dabrisius, J., Lelesius, R., Sereika, V., Zvirbliene, A., Sasnauskas, K., and Gedvilaite, A. (2014). Generation in yeast of recombinant virus-like particles of porcine circovirus type 2 capsid protein and their use for a serologic assay and development of monoclonal antibodies. BMC Biotechnol., 14.","DOI":"10.1186\/s12896-014-0100-1"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"400","DOI":"10.1177\/104063870001200502","article-title":"Development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type 2","volume":"12","author":"Walker","year":"2000","journal-title":"J. Vet. Diagn. Investig."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1186\/s12951-021-00778-8","article-title":"A nanobody-horseradish peroxidase fusion protein-based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2","volume":"19","author":"Mu","year":"2021","journal-title":"J. Nanobiotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"148802","DOI":"10.1016\/j.ijbiomac.2025.148802","article-title":"Sensitive detection of HEV antibodies using a blocking ELISA based on ORF2-specific monoclonal antibodies","volume":"333","author":"Wang","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_12","first-page":"e304024","article-title":"Development and application of a blocking ELISA method based on Cap protein for detecting antibodies against porcine circovirus 2","volume":"13","author":"Liu","year":"2025","journal-title":"Microbiol. Spectr."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.jviromet.2010.09.023","article-title":"Development of a blocking ELISA for detection of serum neutralizing antibodies against porcine circovirus type 2","volume":"171","author":"Huang","year":"2011","journal-title":"J. Virol. Methods"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Lin, C., Li, N., Tao, L., Li, H., Ma, J., Wu, C., Zhang, F., Fang, S., Tan, J., and Huang, Y. (2025). PCV2 Cap protein nuclear import via importin \u03b1\/\u03b2 receptor: Molecular insights and antiviral potential. Front. Microbiol., 16.","DOI":"10.3389\/fmicb.2025.1701697"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s11262-014-1099-z","article-title":"Current understanding of genomic DNA of porcine circovirus type 2","volume":"49","author":"Lv","year":"2014","journal-title":"Virus Genes"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Segal\u00e9s, J., Franzo, G., Larsen, L.E., Nauwynck, H., Opriessnig, T., Stadejek, T., and Vannucci, F. (2025). Knowledge gaps in research and control of porcine circovirus 2 (PCV2) infections. BMC Vet. Res., 22.","DOI":"10.1186\/s12917-025-05134-8"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1016\/j.molimm.2008.10.028","article-title":"Fine mapping of antigenic epitopes on capsid proteins of porcine circovirus, and antigenic phenotype of porcine circovirus type 2","volume":"46","author":"Shang","year":"2009","journal-title":"Mol. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"134955","DOI":"10.1016\/j.ijbiomac.2024.134955","article-title":"Efficacy comparison in cap VLPs of PCV2 and PCV3 as swine vaccine vehicle","volume":"278","author":"Qiu","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"4337","DOI":"10.5455\/OVJ.2025.v15.i9.40","article-title":"Global dynamics of vaccine effectiveness and genotype shift in porcine circovirus 2: A cross-temporal analysis","volume":"15","author":"Orosco","year":"2025","journal-title":"Open Vet. J."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Sagrera, M., Garza-Moreno, L., Cobos, \u00c0., Llorens, A.M., Huerta, E., P\u00e9rez, M., P\u00e9rez, D., Espigares, D., Segal\u00e9s, J., and Sibila, M. (2025). Efficacy of a Novel PCV2d and Mycoplasma hyopneumoniae Combined Vaccine in Piglets with High and Low Levels of PCV2 Maternally Derived Antibodies at Vaccination. Vaccines, 13.","DOI":"10.3390\/vaccines13101076"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Guo, J., Hou, L., Zhou, J., Wang, D., Cui, Y., Feng, X., and Liu, J. (2022). Porcine Circovirus Type 2 Vaccines: Commercial Application and Research Advances. Viruses, 14.","DOI":"10.3390\/v14092005"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e165023","DOI":"10.1128\/jvi.01650-23","article-title":"A novel strategy for an anti-idiotype vaccine: Nanobody mimicking neutralization epitope of porcine circovirus type 2","volume":"98","author":"Deng","year":"2024","journal-title":"J. Virol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e1819","DOI":"10.1128\/JVI.01836-19","article-title":"Neutralization Mechanism of a Monoclonal Antibody Targeting a Porcine Circovirus Type 2 Cap Protein Conformational Epitope","volume":"94","author":"Huang","year":"2020","journal-title":"J. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Sun, M., Wang, S., Fang, Z., Zhao, M., Gao, Y., An, T., Tu, Y., Wang, H., and Cai, X. (2022). A Sandwich ELISA for Quality Control of PCV2 Virus-like Particles Vaccine. Vaccines, 10.","DOI":"10.3390\/vaccines10122175"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2081","DOI":"10.3390\/v15102081","article-title":"Novel Epitope Mapping of African Swine Fever Virus pI215L Protein Using Monoclonal Antibodies","volume":"15","author":"Gao","year":"2023","journal-title":"Viruses"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1016\/j.jviromet.2016.01.011","article-title":"Baculovirus expression of the N-terminus of porcine heat shock protein Gp96 improves the immunogenicity of recombinant PCV2 capsid protein","volume":"230","author":"Zhu","year":"2016","journal-title":"J. Virol. Methods"},{"key":"ref_27","first-page":"58","article-title":"Preparation of monoclonal antibodies against Cap protein of porcine circovirus type 2 and identification of antigenic epitopes","volume":"58","author":"Liu","year":"2026","journal-title":"Anim. Husb. Vet. Med."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"25","DOI":"10.4142\/jvs.2015.16.1.25","article-title":"Assessing PCV2 antibodies in field pigs vaccinated with different porcine circovirus 2 vaccines using two commercial ELISA systems","volume":"16","author":"Shin","year":"2015","journal-title":"J. Vet. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"114954","DOI":"10.1016\/j.jviromet.2024.114954","article-title":"Development a high-sensitivity sandwich ELISA for determining antigen content of porcine circovirus type 2 vaccines","volume":"328","author":"Xu","year":"2024","journal-title":"J. Virol. Methods"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"814","DOI":"10.1016\/j.theriogenology.2007.12.009","article-title":"Prevalence of PCV2 in Austrian and German boars and semen used for artificial insemination","volume":"69","author":"Schmoll","year":"2008","journal-title":"Theriogenology"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3361","DOI":"10.2147\/IJN.S364795","article-title":"Combined Antibody Tagged HRP Gold Nanoparticle Probe for Effective PCV2 Screening in Pig Farms","volume":"17","author":"Zhang","year":"2022","journal-title":"Int. J. Nanomed."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Han, S., Xiao, Y., Zheng, D., Gu, Y., Xuan, Y., Jin, Y., Pang, W., Huang, Y., Li, X., and Deng, J. (2016). Establishment and application of a competitive enzyme-linked immunosorbent assay differentiating PCV2 antibodies from mixture of PCV1\/PCV2 antibodies in pig sera. BMC Vet. Res., 12.","DOI":"10.1186\/s12917-016-0802-9"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1292\/jvms.66.237","article-title":"Development of an ELISA based on the baculovirus-expressed capsid protein of porcine circovirus type 2 as antigen","volume":"66","author":"Liu","year":"2004","journal-title":"J. Vet. Med. Sci."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"274","DOI":"10.1186\/1743-422X-7-274","article-title":"Development and validation of an ELISA using a protein encoded by ORF2 antigenic domain of porcine circovirus type 2","volume":"7","author":"Sun","year":"2010","journal-title":"Virol. J."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3453","DOI":"10.1007\/s00253-019-09715-0","article-title":"A broad spectrum monoclonal antibody against porcine circovirus type 2 for antigen and antibody detection","volume":"103","author":"Huang","year":"2019","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Ding, H., Shen, Y., Gao, Y., Wu, S., Xie, C., Sun, H., Zhang, H., Sun, H., Shan, Y., and Ding, J. (2022). Development of gold Immunochromatographic assay strip based on specific polyclonal antibodies against capsid protein for rapid detection of porcine circovirus 2 in Zhejiang province, China. BMC Vet. Res., 18.","DOI":"10.1186\/s12917-022-03471-6"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"696","DOI":"10.1016\/j.vaccine.2015.09.007","article-title":"Immunogenicity and immunoprotection of porcine circovirus type 2 (PCV2) Cap protein displayed by Lactococcus lactis","volume":"34","author":"Li","year":"2016","journal-title":"Vaccine"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"e35","DOI":"10.4142\/jvs.2019.20.e35","article-title":"Enhanced protective immune response to PCV2 adenovirus vaccine by fusion expression of Cap protein with InvC in pigs","volume":"20","author":"Zhang","year":"2019","journal-title":"J. Vet. Sci."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"4722","DOI":"10.1016\/j.vaccine.2017.07.031","article-title":"Immunogenicity of adenovirus vaccines expressing the PCV2 capsid protein in pigs","volume":"35","author":"Li","year":"2017","journal-title":"Vaccine"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1016\/j.tvjl.2011.08.005","article-title":"Deletion of the single putative N-glycosylation site of the porcine circovirus type 2 Cap protein enhances specific immune responses by DNA immunisation in mice","volume":"192","author":"Gu","year":"2012","journal-title":"Vet. J."}],"container-title":["Veterinary Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2306-7381\/13\/7\/617\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,9]],"date-time":"2026-07-09T04:17:14Z","timestamp":1783570634000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2306-7381\/13\/7\/617"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,25]]},"references-count":40,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["vetsci13070617"],"URL":"https:\/\/doi.org\/10.3390\/vetsci13070617","relation":{},"ISSN":["2306-7381"],"issn-type":[{"value":"2306-7381","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,25]]}}}