{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T12:19:21Z","timestamp":1787746761134,"version":"build-2784847793"},"reference-count":23,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Key Research and Development Project of China","award":["2023YFD1802505"],"award-info":[{"award-number":["2023YFD1802505"]}]},{"name":"National Key Research and Development Project of China","award":["2020YFE0203400"],"award-info":[{"award-number":["2020YFE0203400"]}]},{"name":"Major Science and Technology Program of Xinjiang Uygur Autonomous Region","award":["2024A02005"],"award-info":[{"award-number":["2024A02005"]}]},{"name":"Xinjiang Talent Development Fund","award":["ZZYD2023010"],"award-info":[{"award-number":["ZZYD2023010"]}]},{"name":"Tianchi Talent Introduction Plan","award":["IWA2023"],"award-info":[{"award-number":["IWA2023"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vet. Sci.","Veterinary Sciences"],"abstract":"<jats:p>Strangles, caused by Streptococcus equi subsp. equi (S. equi), is a highly contagious respiratory disease that causes substantial economic losses to the horse industry worldwide. Serological diagnosis currently relies mainly on the imported IDvet\u00ae dual-antigen sandwich ELISA kit, which shows good diagnostic performance but is expensive and lacks a domestic alternative in China, thereby limiting its routine use in primary veterinary settings. The aim of this study was to develop a domestic indirect ELISA based on novel antigen targets and an untagged antigen purification strategy. Using comparative bioinformatics analyses, two conserved fragments with species-level specificity for S. equi, designated EQ8 (150 aa) and SclF (44 aa), were identified and fused for recombinant expression. The fusion protein was purified by GST affinity chromatography, cleaved with PreScission protease, and subjected to secondary purification to obtain an untagged EQ8\u2013SclF protein. Western blotting and ELISA demonstrated that the purified protein retained good immunoreactivity and that the GST tag did not cause detectable cross-reactivity, with all OD450 values obtained using GST-coated plates remaining below 0.09, well under the cut-off value of 0.115. Checkerboard titration established the optimal assay conditions as an antigen coating concentration of 1 \u03bcg\/mL and a serum dilution of 1:100. Using the IDvet kit as the reference method, 157 clinical serum samples (67 positive and 90 negative) were tested in parallel. The newly developed assay showed a relative sensitivity of 89.55% (60\/67), a relative specificity of 94.44% (85\/90), an overall agreement rate of 92.36% (145\/157), and a kappa value of 0.84, indicating excellent agreement between the two methods. Good inter-assay reproducibility was observed, with coefficients of variation all below 9.29%. In conclusion, an indirect ELISA based on an untagged EQ8\u2013SclF fusion protein was preliminarily established for the serological detection of strangles. This assay showed diagnostic performance highly consistent with that of the imported commercial kit while offering potential advantages in terms of lower cost, simpler operation, and elimination of tag-associated interference. It therefore shows potential as a domestic alternative for the serological diagnosis of strangles.<\/jats:p>","DOI":"10.3390\/vetsci13090866","type":"journal-article","created":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T10:59:23Z","timestamp":1787741963000},"page":"866","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development and Evaluation of an Indirect ELISA for Strangles Based on an Untagged EQ8\u2013SclF Fusion Protein"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-7166-816X","authenticated-orcid":false,"given":"Rongkuan","family":"Sun","sequence":"first","affiliation":[{"name":"College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"},{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haoyu","family":"Zu","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1507-737X","authenticated-orcid":false,"given":"Wei","family":"Guo","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"},{"name":"China-Kazakhstan Joint Laboratory for Herbivorous Animal Disease Research, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"},{"name":"Institute of Western Agriculture, Chinese Academy of Agricultural Sciences, Changji 831100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4521-4099","authenticated-orcid":false,"given":"Xiaojun","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"},{"name":"China-Kazakhstan Joint Laboratory for Herbivorous Animal Disease Research, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"},{"name":"Institute of Western Agriculture, Chinese Academy of Agricultural Sciences, Changji 831100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2349-0028","authenticated-orcid":false,"given":"Qian","family":"Yang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"633","DOI":"10.1111\/jvim.15043","article-title":"Streptococcus equi Infections in Horses: Guidelines for Treatment, Control, and Prevention of Strangles-Revised Consensus Statement","volume":"32","author":"Boyle","year":"2018","journal-title":"J. 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