{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T11:18:58Z","timestamp":1788347938602,"version":"build-2803163510"},"reference-count":25,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,17]],"date-time":"2026-01-17T00:00:00Z","timestamp":1768608000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Innovation Program of the Chinese Academy of Agricultural Sciences","award":["CAAS-CSLPDCP-202402"],"award-info":[{"award-number":["CAAS-CSLPDCP-202402"]}]},{"DOI":"10.13039\/501100012453","name":"China Agriculture Research System","doi-asserted-by":"publisher","award":["CARS-41-G15"],"award-info":[{"award-number":["CARS-41-G15"]}],"id":[{"id":"10.13039\/501100012453","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Reticuloendotheliosis virus (REV) is an immunosuppressive virus in poultry that can cause acute reticular neoplasms, chronic lymphoid tumors, stunting syndrome, and secondary infections. In many countries, the lack of effective vaccines has resulted in a high prevalence of REV infections and substantial economic losses. Enzyme-linked immunosorbent assay (ELISA)-based antibody detection is an important tool for monitoring the REV prevalence in poultry farms. ELISA coating antigens generally consist of either whole virus or viral protein; however, most commercially available REV antibody ELISA detection kits use whole virus as the coating antigen, which limits their applicability in certain diagnostic and research settings. In this study, the gp90 protein from a dominant REV strain was expressed and purified using 293F suspension cell eukaryotic expression system. Using recombinant gp90 protein as the coating antigen, an indirect ELISA for detecting gp90 antibodies (gp90-ELISA) was developed. After optimization, the optimal conditions were as follows: coating antigen concentration of 4 \u00b5g\/mL with overnight incubation at 4 \u00b0C; blocking with 5% skim milk at 37 \u00b0C for 1.5 h; serum dilution of 1:200 with incubation at 37 \u00b0C for 45 min; secondary antibody dilution of 1:1000 with incubation at 37 \u00b0C for 30 min; and color development using TMB substrate at room temperature in the dark for 10 min. The cut-off value was defined as an OD450 \u2265 0.22 for positive samples and &lt;0.22 for negative samples. The developed gp90-ELISA specifically detected REV-positive sera at a maximum serum dilution ratio of 1:3200. Intra- and inter-assay variation coefficients were \u226410%, indicating that the gp90-ELISA had good specificity, sensitivity, and reproducibility. Laboratory serum testing showed that the gp90-ELISA successfully detected sera from chickens immunized with the gp90 protein or infected with REV. Furthermore, analysis of clinical serum samples demonstrated 100% concordance between the gp90-ELISA results and a commercial whole-virus-coated ELISA kit. These results indicate that the gp90-ELISA is a reliable supplementary method to whole-virus-coated ELISA and has potential utility in disease surveillance and evaluation of immune responses.<\/jats:p>","DOI":"10.3390\/v18010124","type":"journal-article","created":{"date-parts":[[2026,1,19]],"date-time":"2026-01-19T08:23:56Z","timestamp":1768811036000},"page":"124","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Development of an Indirect ELISA for REV gp90 Antibody Detection Using the gp90 Protein Expressed in Suspended Cells"],"prefix":"10.3390","volume":"18","author":[{"given":"Erjing","family":"Ke","sequence":"first","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"},{"name":"Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300392, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengmeng","family":"Huang","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guodong","family":"Wang","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingzhe","family":"Han","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"},{"name":"Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300392, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yulong","family":"Zhang","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Runhang","family":"Liu","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hangbo","family":"Yu","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziwen","family":"Wu","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dan","family":"Ling","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xianyun","family":"Liu","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"},{"name":"Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300392, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tengfei","family":"Xu","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suyan","family":"Wang","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuntong","family":"Chen","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongzhen","family":"Liu","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanping","family":"Zhang","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongyu","family":"Cui","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yulu","family":"Duan","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liuan","family":"Li","sequence":"additional","affiliation":[{"name":"Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300392, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8570-823X","authenticated-orcid":false,"given":"Xiaoxue","family":"Yu","sequence":"additional","affiliation":[{"name":"Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300392, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0448-8467","authenticated-orcid":false,"given":"Yulong","family":"Gao","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"},{"name":"Heilongjiang Province Key Laboratory of Veterinary Immunology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"},{"name":"Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonosis, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaole","family":"Qi","sequence":"additional","affiliation":[{"name":"Avian Immunosuppressive Diseases Division, State Key Laboratory of Animal Disease 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(2015). Transcriptional pofiling of host gene expression in chicken embryo fibroblasts infected with reticuloendotheliosis virus strain HA1101. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0126992"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1187","DOI":"10.1007\/s00705-016-3180-5","article-title":"Analysis of the spleen proteome of chickens infected with reticuloendotheliosis virus","volume":"162","author":"Xue","year":"2017","journal-title":"Arch. Virol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Chac\u00f3n, R.D., Sedano-Herrera, B., Alfaro-Espinoza, E.R., Quispe, W.U., Li\u00f1an-Torres, A., De la Torre, D., de Oliveira, A., Astolfi-Ferreira, C.S., and Ferreira, A.J.P. (2022). Complete genome characterization of reticuloendotheliosis virus detected in chickens with multiple viral coinfections. Viruses, 14.","DOI":"10.3390\/v14040798"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"334","DOI":"10.1080\/03079457.2024.2435887","article-title":"The complexity of the interpretation of ELISA and RT-PCR results in the diagnosis of a reticuloendotheliosis virus infection: An extended case study","volume":"54","author":"Mamczur","year":"2025","journal-title":"Avian Pathol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"139","DOI":"10.7589\/JWD-D-23-00012","article-title":"Lymphoproliferative disease virus and reticuloendotheliosis virus detection and disease in wild turkeys (Meleagris gallopavo)","volume":"60","author":"Adcock","year":"2024","journal-title":"J. Wildl. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"257","DOI":"10.2478\/jvetres-2018-0036","article-title":"Avian reticuloendotheliosis in chickens\u2014An update on disease occurrence and clinical course","volume":"62","author":"Frant","year":"2018","journal-title":"Vet. Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1186\/s12985-024-02525-5","article-title":"Molecular characterization and immunopathological investigation of Avian reticuloendotheliosis virus in breeder flocks in Egypt","volume":"21","author":"Shosha","year":"2024","journal-title":"Virol. J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1186\/s12985-018-0948-0","article-title":"Protection induced by a gp90 protein-based vaccine derived from a reticuloendotheliosis virus strain isolated from a contaminated IBD vaccine","volume":"15","author":"Ren","year":"2018","journal-title":"Virol. J."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1944","DOI":"10.1016\/j.vaccine.2013.02.010","article-title":"Enhancement of humoral and cellular immunity in chickens against reticuloendotheliosis virus by DNA prime-protein boost vaccination","volume":"31","author":"Li","year":"2013","journal-title":"Vaccine"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5653","DOI":"10.1016\/j.vaccine.2007.02.053","article-title":"Recombinant viral proteins for use in diagnostic ELISAs to detect virus infection","volume":"25","author":"Spencer","year":"2007","journal-title":"Vaccine"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1637\/10290-062512-Reg.1","article-title":"Detection of anti-reticuloendotheliosis virus antibody by blocking enzyme-linked immunosorbent assay with expression envelope protein","volume":"57","author":"Wu","year":"2013","journal-title":"Avian Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.antiviral.2017.04.019","article-title":"Immunoprotection induced by CpG-ODN\/Poly(I:C) combined with recombinant gp90 protein in chickens against reticuloendotheliosis virus infection","volume":"147","author":"Yuan","year":"2017","journal-title":"Antivir. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1128\/jvi.63.1.273-280.1989","article-title":"Role of reticuloendotheliosis virus envelope glycoprotein in superinfection interference","volume":"63","author":"Delwart","year":"1989","journal-title":"J. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Xue, M., Shi, X., Zhang, J., Zhao, Y., Cui, H., Hu, S., Gao, H., Cui, X., and Wang, Y.F. (2012). Identification of a conserved B-cell epitope on reticuloendotheliosis virus envelope protein by screening a phage-displayed random peptide library. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0049842"},{"key":"ref_15","first-page":"23","article-title":"Isolation, identification and in vitro replication of reticuloendotheliosis virus in chickens","volume":"18","author":"Deng","year":"2010","journal-title":"Chin. J. Anim. Infect. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1016\/j.virusres.2012.02.011","article-title":"Construction and characterization of the infectious clone of Reticuloendotheliosis virus carrying a genetic marker","volume":"167","author":"Deng","year":"2012","journal-title":"Virus Res."},{"key":"ref_17","first-page":"66","article-title":"Preparation and preliminary application of monoclonal antibodies against the envelope protein of avian reticuloendotheliosis virus","volume":"47","author":"Han","year":"2025","journal-title":"Chin. Poult."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.jviromet.2011.12.008","article-title":"Development of an indirect ELISA for serological detection of reticuloendotheliosis virus using the gp90 protein expressed in Pichia pastoris","volume":"180","author":"Li","year":"2012","journal-title":"J. Virol. Methods"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"S6","DOI":"10.2460\/javma.24.10.0657","article-title":"The diagnostic process for infectious disease diagnosis in animals","volume":"263","author":"Burrough","year":"2024","journal-title":"J. Am. Vet. Med. Assoc."},{"key":"ref_20","first-page":"107","article-title":"Development of an indirect ELISA method for detecting antibodies against avian reticuloendotheliosis virus based on the p30-gp90 fusion protein","volume":"38","author":"Luan","year":"2021","journal-title":"Chin. J. Anim. Quar."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"160196","DOI":"10.1098\/rsob.160196","article-title":"High-throughput recombinant protein expression in Escherichia coli: Current status and future perspectives","volume":"6","author":"Jia","year":"2016","journal-title":"Open Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1007\/978-1-59745-209-0_2","article-title":"Cloning, production, and purification of proteins for a medium-scale structural genomics project","volume":"363","author":"Collinet","year":"2007","journal-title":"Methods Mol. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Choi, Y.S., Yoon, S., Kim, K.L., Yoo, J., Song, P., Kim, M., Shin, Y.E., Yang, W.J., Noh, J.E., and Cho, H.S. (2014). Computational design of binding proteins to EGFR domain II. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0092513"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1140","DOI":"10.2307\/1590468","article-title":"Epidemiology of reticuloendotheliosis virus in broiler breeder flocks","volume":"29","author":"Witter","year":"1985","journal-title":"Avian Dis."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1007\/BF01310977","article-title":"Serologic differences among nondefective reticuloendotheliosis viruses","volume":"93","author":"Chen","year":"1987","journal-title":"Arch. Virol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/1\/124\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,21]],"date-time":"2026-01-21T11:14:19Z","timestamp":1768994059000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/1\/124"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,17]]},"references-count":25,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["v18010124"],"URL":"https:\/\/doi.org\/10.3390\/v18010124","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,17]]}}}