{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T01:30:34Z","timestamp":1792027834399,"version":"4.4.0"},"reference-count":36,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2021,11,30]],"date-time":"2021-11-30T00:00:00Z","timestamp":1638230400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Porcine deltacoronavirus (PDCoV) can cause diarrhea and dehydration in newborn piglets. Here, we developed a double antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-ELISA) for detection of PDCoV by using a specific monoclonal antibody against the PDCoV N protein and an anti-PDCoV rabbit polyclonal antibody. Using DAS-ELISA, the detection limit of recombinant PDCoV N protein and virus titer were approximately 0.5 ng\/mL and 103.0 TCID50\/mL, respectively. A total of 59 intestinal and 205 fecal samples were screened for the presence of PDCoV by using DAS-ELISA and reverse transcriptase real-time PCR (RT-qPCR). The coincidence rate of the DAS-ELISA and RT-qPCR was 89.8%. DAS-ELISA had a sensitivity of 80.8% and specificity of 95.6%. More importantly, the DAS-ELISA could detect the antigen of PDCoV inactivated virus, and the viral antigen concentrations remained unchanged in the inactivated virus. These results suggest that DAS-ELISA could be used for antigen detection of clinical samples and inactivated vaccines. It is a novel method for detecting PDCoV infections and evaluating the PDCoV vaccine.<\/jats:p>","DOI":"10.3390\/v13122403","type":"journal-article","created":{"date-parts":[[2021,12,1]],"date-time":"2021-12-01T03:12:56Z","timestamp":1638328376000},"page":"2403","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":37,"title":["Development of a Novel Double Antibody Sandwich ELISA for Quantitative Detection of Porcine Deltacoronavirus Antigen"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0721-447X","authenticated-orcid":false,"given":"Wei","family":"Wang","sequence":"first","affiliation":[{"name":"Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology Ministry of Agriculture, Nanjing 210014, China"},{"name":"Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing 210014, China"},{"name":"Jiangsu Co-Infection Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China"},{"name":"Jiangsu Key Laboratory of Zoonoses, Yangzhou University, Yangzhou 225000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jizong","family":"Li","sequence":"additional","affiliation":[{"name":"Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology Ministry of Agriculture, Nanjing 210014, China"},{"name":"Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing 210014, China"},{"name":"Jiangsu Co-Infection Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China"},{"name":"Jiangsu Key Laboratory of Zoonoses, Yangzhou University, Yangzhou 225000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Baochao","family":"Fan","sequence":"additional","affiliation":[{"name":"Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology Ministry of Agriculture, Nanjing 210014, China"},{"name":"Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing 210014, China"},{"name":"Jiangsu Co-Infection Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China"},{"name":"Jiangsu Key Laboratory of Zoonoses, Yangzhou University, Yangzhou 225000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuehan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology Ministry of Agriculture, Nanjing 210014, China"},{"name":"Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing 210014, China"},{"name":"Jiangsu Co-Infection Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China"},{"name":"Jiangsu Key Laboratory of Zoonoses, Yangzhou University, Yangzhou 225000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rongli","family":"Guo","sequence":"additional","affiliation":[{"name":"Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology Ministry of Agriculture, Nanjing 210014, China"},{"name":"Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing 210014, China"},{"name":"Jiangsu Co-Infection Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China"},{"name":"Jiangsu Key Laboratory of Zoonoses, Yangzhou University, Yangzhou 225000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongxiang","family":"Zhao","sequence":"additional","affiliation":[{"name":"Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology Ministry of Agriculture, Nanjing 210014, China"},{"name":"Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing 210014, China"},{"name":"Jiangsu Co-Infection Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China"},{"name":"Jiangsu Key Laboratory of Zoonoses, Yangzhou University, Yangzhou 225000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junming","family":"Zhou","sequence":"additional","affiliation":[{"name":"Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology Ministry of Agriculture, Nanjing 210014, China"},{"name":"Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing 210014, China"},{"name":"Jiangsu Co-Infection Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China"},{"name":"Jiangsu Key Laboratory of Zoonoses, Yangzhou University, Yangzhou 225000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinzhu","family":"Zhou","sequence":"additional","affiliation":[{"name":"Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology Ministry of Agriculture, Nanjing 210014, China"},{"name":"Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing 210014, China"},{"name":"Jiangsu Co-Infection Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China"},{"name":"Jiangsu Key Laboratory of Zoonoses, Yangzhou University, Yangzhou 225000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dongbo","family":"Sun","sequence":"additional","affiliation":[{"name":"Laboratory for the Prevention and Control of Swine Infectious Diseases, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1318-7081","authenticated-orcid":false,"given":"Bin","family":"Li","sequence":"additional","affiliation":[{"name":"Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology Ministry of Agriculture, Nanjing 210014, China"},{"name":"Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing 210014, China"},{"name":"Jiangsu Co-Infection Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China"},{"name":"Jiangsu Key Laboratory of Zoonoses, Yangzhou University, Yangzhou 225000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,11,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/j.meegid.2019.04.023","article-title":"Detection and spike gene characterization in porcine deltacoronavirus in China during 2016\u20132018","volume":"73","author":"Zhang","year":"2019","journal-title":"Infect. Genet. Evol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1347","DOI":"10.3201\/eid2008.140526","article-title":"Rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus","volume":"20","author":"Marthaler","year":"2014","journal-title":"Emerg. Infect. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.virusres.2016.04.009","article-title":"Porcine deltacoronavirus infection: Etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis","volume":"226","author":"Jung","year":"2016","journal-title":"Virus Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3995","DOI":"10.1128\/JVI.06540-11","article-title":"Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus","volume":"86","author":"Woo","year":"2012","journal-title":"J. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1227","DOI":"10.3201\/eid2007.140296","article-title":"Detection and Genetic Characterization of Deltacoronavirus in Pigs, Ohio, USA, 2014","volume":"20","author":"Wang","year":"2014","journal-title":"Emerg. Infect. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1594","DOI":"10.3201\/eid2009.140756","article-title":"Porcine coronavirus HKU15 detected in 9 US states, 2014","volume":"20","author":"Wang","year":"2014","journal-title":"Emerg. Infect. Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"757","DOI":"10.3201\/eid2204.151852","article-title":"Porcine Deltacoronavirus, Thailand, 2015","volume":"22","author":"Janetanakit","year":"2016","journal-title":"Emerg. Infect. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1111\/tbed.12490","article-title":"Detection and Phylogenetic Analysis of Porcine Deltacoronavirus in Korean Swine Farms, 2015","volume":"63","author":"Lee","year":"2016","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1197","DOI":"10.1111\/tbed.12858","article-title":"Herd-level prevalence and incidence of porcine epidemic diarrhoea virus (PEDV) and porcine deltacoronavirus (PDCoV) in swine herds in Ontario, Canada","volume":"65","author":"Ajayi","year":"2018","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2909","DOI":"10.1007\/s00705-016-2983-8","article-title":"The first detection and full-length genome sequence of porcine deltacoronavirus isolated in Lao PDR","volume":"161","author":"Lorsirigool","year":"2016","journal-title":"Arch. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1016\/j.meegid.2018.03.030","article-title":"Genetic characterization and pathogenicity of Japanese porcine deltacoronavirus","volume":"61","author":"Suzuki","year":"2018","journal-title":"Infect. Genet. Evol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e00237-17","DOI":"10.1128\/genomeA.00237-17","article-title":"Complete Genome Characterization of Chinese Porcine Deltacoronavirus Strain CHN\/Tianjin\/2016","volume":"5","author":"Zhao","year":"2017","journal-title":"Genome Announc."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1016\/j.virusres.2016.05.028","article-title":"Porcine deltacoronavirus: Overview of infection dynamics, diagnostic methods, prevalence and genetic evolution","volume":"226","author":"Zhang","year":"2016","journal-title":"Virus Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2975","DOI":"10.1007\/s00705-021-05226-4","article-title":"Porcine deltacoronavirus and its prevalence in China: A review of epidemiology, evolution, and vaccine development","volume":"166","author":"Tang","year":"2021","journal-title":"Arch. Virol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"678","DOI":"10.1111\/tbed.13385","article-title":"Development of a multiplex RT-PCR for the detection of major diarrhoeal viruses in pig herds in China","volume":"67","author":"Ding","year":"2020","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"114096","DOI":"10.1016\/j.jviromet.2021.114096","article-title":"Rapid detection of porcine deltacoronavirus and porcine epidemic diarrhea virus using the duplex recombinase polymerase amplification method","volume":"292","author":"Li","year":"2021","journal-title":"J. Virol. Methods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1080\/21505594.2020.1771980","article-title":"Development of a TaqMan-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses","volume":"11","author":"Pan","year":"2020","journal-title":"Virulence"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"540248","DOI":"10.3389\/fvets.2020.540248","article-title":"Development of a Novel Double Antibody Sandwich Quantitative Enzyme-Linked Immunosorbent Assay for Detection of Porcine Epidemic Diarrhea Virus Antigen","volume":"7","author":"Fan","year":"2020","journal-title":"Front. Vet. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1089\/mab.2013.0023","article-title":"Monoclonal antibodies against Nsp2 protein of the highly pathogenic porcine reproductive and respiratory syndrome virus","volume":"32","author":"Li","year":"2013","journal-title":"Monoclon. Antibodies Immunodiagn. Immunother."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1084","DOI":"10.1177\/22.12.1084","article-title":"Peroxidase-labeled antibody. A new method of conjugation","volume":"22","author":"Nakane","year":"1974","journal-title":"J. Histochem. Cytochem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1128\/CVI.00355-10","article-title":"Differentiation between human coronaviruses NL63 and 229E using a novel double-antibody sandwich enzyme-linked immunosorbent assay based on specific monoclonal antibodies","volume":"18","author":"Sastre","year":"2011","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_22","first-page":"62","article-title":"Kappa coefficient: A popular measure of rater agreement","volume":"27","author":"Tang","year":"2015","journal-title":"Shanghai Arch. Psychiatry"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"159","DOI":"10.2307\/2529310","article-title":"The measurement of observer agreement for categorical data","volume":"33","author":"Landis","year":"1977","journal-title":"Biometrics"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.virol.2015.03.024","article-title":"Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets","volume":"482","author":"Chen","year":"2015","journal-title":"Virology"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"650","DOI":"10.3201\/eid2104.141859","article-title":"Pathogenicity of 2 Porcine Deltacoronavirus Strains in Gnotobiotic Pigs","volume":"21","author":"Jung","year":"2015","journal-title":"Emerg. Infect. Dis."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e00064-15","DOI":"10.1128\/mBio.00064-15","article-title":"Origin, evolution, and virulence of porcine deltacoronaviruses in the United States","volume":"6","author":"Ma","year":"2015","journal-title":"MBio"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e00218-14","DOI":"10.1128\/genomeA.00218-14","article-title":"Complete Genome Sequence of Strain SDCV\/USA\/Illinois121\/2014, a Porcine Deltacoronavirus from the United States","volume":"2","author":"Marthaler","year":"2014","journal-title":"Genome Announc."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.jviromet.2016.03.016","article-title":"Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus","volume":"234","author":"Zhang","year":"2016","journal-title":"J. Virol. Methods"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Thachil, A., Gerber, P.F., Xiao, C.T., Huang, Y.W., and Opriessnig, T. (2015). Development and application of an ELISA for the detection of porcine deltacoronavirus IgG antibodies. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0124363"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1292\/jvms.15-0533","article-title":"A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus","volume":"78","author":"Su","year":"2016","journal-title":"J. Vet. Med. Sci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.vetmic.2016.02.004","article-title":"Two-way antigenic cross-reactivity between porcine epidemic diarrhea virus and porcine deltacoronavirus","volume":"186","author":"Ma","year":"2016","journal-title":"Vet. Microbiol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1016\/j.rvsc.2009.05.009","article-title":"Comparison of enzyme-linked immunosorbent assay and RT-PCR for the detection of porcine epidemic diarrhoea virus","volume":"88","author":"Sozzi","year":"2010","journal-title":"Res. Vet. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1016\/j.exppara.2011.10.012","article-title":"A double antibody sandwich enzyme-linked immunosorbent assay for detection of secreted antigen 1 of Babesia microti using hamster model","volume":"130","author":"Luo","year":"2012","journal-title":"Exp. Parasitol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.jviromet.2005.08.009","article-title":"Indirect sandwich ELISA for antigen detection of African swine fever virus: Comparison of polyclonal and monoclonal antibodies","volume":"131","author":"Hutchings","year":"2006","journal-title":"J. Virol. Methods"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1016\/j.fsi.2009.11.015","article-title":"Immune response of Japanese flounder Paralichthys olivaceus to outer membrane protein of Edwardsiella tarda","volume":"28","author":"Tang","year":"2010","journal-title":"Fish Shellfish. Immunol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1016\/j.jviromet.2017.02.003","article-title":"Development of a monoclonal sandwich ELISA for direct detection of bluetongue virus 8 in infected animals","volume":"243","author":"Kohl","year":"2017","journal-title":"J. Virol. Methods"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/12\/2403\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:38:07Z","timestamp":1760168287000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/12\/2403"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,11,30]]},"references-count":36,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2021,12]]}},"alternative-id":["v13122403"],"URL":"https:\/\/doi.org\/10.3390\/v13122403","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,11,30]]}}}