Veterinary sciences2026; 13(9); 866; doi: 10.3390/vetsci13090866

Development and Evaluation of an Indirect ELISA for Strangles Based on an Untagged EQ8-SclF Fusion Protein.

Sun, R
·
Zu, H
·
Wang, M
·
Guo, W
·
Wang, X
·
Yang, Q

Abstract: Strangles, caused by subsp. (), 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 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 , 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-SclF 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 OD 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 μg/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-SclF 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.

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Publication Date: 2026-08-26 PubMed ID: 42797902PubMed Central: PMC13611558DOI: 10.3390/vetsci13090866Google Scholar: Lookup

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Summary
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Conflict of Interest
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Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

This study developed and validated a new, cost-effective indirect ELISA test for detecting strangles, a contagious horse respiratory disease, using a novel untagged fusion protein as antigen.
The newly developed ELISA showed comparable diagnostic accuracy to an existing imported commercial test, offering a promising domestic alternative for routine veterinary use in China.

Background and Problem Addressed

Strangles is a highly contagious respiratory disease in horses caused by the bacterium Streptococcus equi subsp. equi, leading to significant economic losses globally.
Serological diagnosis of strangles primarily relies on an imported IDvet dual-antigen sandwich ELISA kit.
Limitations of the existing commercial kit include high cost and lack of domestic alternatives in China, restricting its widespread use, especially in primary veterinary settings.

Objectives

Develop a domestic, indirect ELISA test for strangles detection using novel antigen targets derived from conserved bacterial protein fragments.
Establish an untagged antigen purification strategy to avoid potential assay interference linked to affinity tags.
Evaluate the diagnostic performance of the newly developed ELISA against the commercial IDvet kit.

Antigen Design and Protein Purification

Bioinformatics analyses identified two conserved fragments specific to S. equi: EQ8 (150 amino acids) and SclF (44 amino acids).
These fragments were genetically fused to create a recombinant fusion protein for the ELISA antigen.
The fusion protein was initially purified using GST affinity chromatography.
To remove the GST tag and avoid cross-reactivity, the fusion protein was cleaved with PreScission protease.
Secondary purification steps yielded an untagged EQ8-SclF protein for use as the ELISA antigen.

Validation of Antigen Immunoreactivity

Western blotting and ELISA tests confirmed that the untagged EQ8-SclF protein maintained strong immunoreactivity.
GST tag presence did not cause significant cross-reactivity, as demonstrated by low optical density (OD) values (<0.09) on GST-coated plates, well below the cut-off (0.115).

Optimization of ELISA Assay Conditions

Checkerboard titration was used to determine the optimal ELISA setup.
Optimal antigen coating concentration was established at 1 μg/mL.
Optimal serum dilution was set at 1:100.

Diagnostic Performance Evaluation

157 clinical serum samples were tested in parallel using both the new ELISA and the IDvet commercial kit (67 positive and 90 negative samples).
Relative sensitivity of the new assay was 89.55% (detected 60/67 positives), indicating its ability to correctly identify infected cases.
Relative specificity was 94.44% (correctly identified 85/90 negatives), reflecting low false positives.
Overall agreement rate between the two methods was 92.36% (145/157 samples consistent results).
Kappa value of 0.84 indicated excellent concordance between new and commercial ELISA tests.

Reproducibility and Practical Implications

Inter-assay reproducibility was strong, with coefficients of variation all below 9.29%, underscoring assay reliability.
The newly developed ELISA is simpler to operate, more cost-effective, and eliminates interference caused by affinity tags used in antigen purification.
This assay represents a promising domestic alternative for serological diagnosis of strangles in China, facilitating broader access in veterinary clinics and improving disease management.

Conclusions

The study successfully established an indirect ELISA using an untagged EQ8-SclF fusion protein as antigen for strangles diagnosis.
The new test demonstrated diagnostic performance comparable to the imported IDvet kit.
Advantages include lower cost, ease of operation, and avoidance of tag-related cross-reactions.
Overall, the assay shows strong potential for adoption as a routine diagnostic tool in domestic veterinary settings.

Cite This Article

APA

Sun R, Zu H, Wang M, Guo W, Wang X, Yang Q. (2026). Development and Evaluation of an Indirect ELISA for Strangles Based on an Untagged EQ8-SclF Fusion Protein. Vet Sci, 13(9), 866. https://doi.org/10.3390/vetsci13090866

Publication

Veterinary sciences

ISSN: 2306-7381

NlmUniqueID: 101680127

Country: Switzerland

Language: English

Volume: 13

Issue: 9

PII: 866

Researcher Affiliations

Sun, Rongkuan

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.

Zu, Haoyu

State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.

Wang, Min

State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.

Guo, Wei

State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
China-Kazakhstan Joint Laboratory for Herbivorous Animal Disease Research, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Institute of Western Agriculture, Chinese Academy of Agricultural Sciences, Changji 831100, China.

Wang, Xiaojun

State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
China-Kazakhstan Joint Laboratory for Herbivorous Animal Disease Research, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Institute of Western Agriculture, Chinese Academy of Agricultural Sciences, Changji 831100, China.

Yang, Qian

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.

Grant Funding

2023YFD1802505 & 2020YFE0203400 / the National Key Research and Development Project of China
2024A02005 / the Major Science and Technology Program of Xinjiang Uygur Autonomous Region
ZZYD2023010 / the Xinjiang Talent Development Fund
IWA2023 / the Tianchi Talent Introduction Plan

Conflict of Interest Statement

The authors declare no conflicts of interest.

References

This article includes 23 references

Boyle AG, Timoney JF, Newton JR, Hines MT, Waller AS, Buchanan BR. Streptococcus equi Infections in Horses: Guidelines for Treatment, Control, and Prevention of Strangles-Revised Consensus Statement.. J. Vet. Intern. Med. 2018;32:633–647.
doi: 10.1111/jvim.15043pmc: PMC5867011pubmed: 29424487google scholar: lookup
Latif S, Yousif A. Molecular Study of Streptococcus equi Isolated from Horses with Strangles in Iraq.. Open Vet. J. 2025;15:731–737.
doi: 10.5455/ovj.2025.v15.i2.22pmc: PMC11974311pubmed: 40201849google scholar: lookup
Lee H, Yun SH, Hyon J, Lee S-Y, Yi Y-S, Choi C-W, Jun S, Park EC, Kim SI. Dataset of Single Nucleotide Polymorphisms and Comprehensive Proteomic Analysis of Streptococcus equi Subsp. equi ATCC 39506.. Data Brief. 2021;38:107402.
doi: 10.1016/j.dib.2021.107402pmc: PMC8479396pubmed: 34621931google scholar: lookup
Slater JD. Strangles, Bastard Strangles, Vives and Glanders: Archaeological Relics in a Genomic Age.. Equine Vet. J. 2010;35:118–120.
doi: 10.2746/042516403776114252pubmed: 12638785google scholar: lookup
Tharwat M, Sobayil F. Equine Colic: A Comprehensive Overview of the Sonographic Evaluation, Diagnostic Criteria, and Management of Different Categories.. Open Vet. J. 2025;15:1116–1139.
doi: 10.5455/ovj.2025.v15.i3.5pmc: PMC12017722pubmed: 40276205google scholar: lookup
North SE, Wakeley PR, Mayo N, Mayers J, Sawyer J. Development of a Real-Time PCR to Detect Streptococcus equi subspecies equi.. Equine Vet. J. 2013;46:56–59.
doi: 10.1111/evj.12088pubmed: 23663066google scholar: lookup
Katy W, Colin B, Tihana H, Zoe H, Karen FS, Carl R, J Richard N, Andrew SW. Detection of Streptococcus equi Subspecies equi Using a Triplex QPCR Assay.. Vet. J. 2013;195:300–304.
doi: 10.1016/j.tvjl.2012.07.007pmc: PMC3611602pubmed: 22884566google scholar: lookup
Ranganathan A, Paradise GA, Hansen CA, McCoy MR, Gee SJ, Zhong P, Chang D, Hammock BD. Indirect Homologous Competitive Enzyme-Linked Immunosorbent Assay for the Detection of a Class of Glycosylated Dihydrochalcones.. J. Agric. Food Chem. 2013;61:6964–6970.
doi: 10.1021/jf401302ypmc: PMC3723731pubmed: 23767873google scholar: lookup
Robinson C, Steward KF, Potts N, Barker C, Hammond T, Pierce K, Gunnarsson E, Svansson V, Slater J, Newton JR. Combining Two Serological Assays Optimises Sensitivity and Specificity for the Identification of Streptococcus equi Subsp. equi Exposure.. Vet. J. 2013;197:188–191.
doi: 10.1016/j.tvjl.2013.01.033pubmed: 23465547google scholar: lookup
Wan J, Weldon E, Ganser G, Ruth E, Hughes EV, Bordin AI, Heine PA, Hust M, Cohen ND, Gill JJ. Immunogenic Streptococcus equi Cell Surface Proteins Identified by ORFeome Phage Display.. mSphere. 2025;10:e0062625.
doi: 10.1128/msphere.00626-25pmc: PMC12724188pubmed: 41288106google scholar: lookup
Frosth S, Ruth E, Wilson H, Frykberg L, Jacobsson K, Parkhill J, Flock J, Wood T, Guss B, Aanensen DM. Conservation of Vaccine Antigen Sequences Encoded by Sequenced Strains of Streptococcus equi Subsp. equi.. Equine Vet. J. 2022;55:92–101.
doi: 10.1111/evj.13552pmc: PMC10078666pubmed: 35000217google scholar: lookup
Landis RJ, Koch GG. The Measurement of Observer Agreement for Categorical Data.. Biometrics. 1977;33:159–174.
doi: 10.2307/2529310pubmed: 843571google scholar: lookup
Muller JD, Wilkins M, Foord AJ, Dolezal O, Yu M, Heine HG, Wang L-F. Improvement of a Recombinant Antibody-Based Serological Assay for Foot-And-Mouth Disease Virus.. J. Immunol. Methods. 2010;352:81–88.
doi: 10.1016/j.jim.2009.11.004pmc: PMC7094511pubmed: 19913020google scholar: lookup
Andreolla AP, Marina L, Frandoloso R, Luiz Carlos Kreutz. Development of an Indirect ELISA Based on Recombinant Capsid Protein to Detect Antibodies to Bovine Leukemia Virus.. Braz. J. Microbiol. 2018;49:68–75.
doi: 10.1016/j.bjm.2018.05.001pmc: PMC6328717pubmed: 29866609google scholar: lookup
Tiwari P, Kaila P, Guptasarma P. Understanding Anomalous Mobility of Proteins on SDS-PAGE with Special Reference to the Highly Acidic Extracellular Domains of Human E- and N-Cadherins.. ELECTROPHORESIS. 2019;40:1273–1281.
doi: 10.1002/elps.201800219pubmed: 30702765google scholar: lookup
Graceffa P, Jancsó A, Mabuchi K. Modification of Acidic Residues Normalizes Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis of Caldesmon and Other Proteins That Migrate Anomalously.. Arch. Biochem. Biophys. 1992;297:46–51.
doi: 10.1016/0003-9861(92)90639-epubmed: 1637182google scholar: lookup
Shi X-Z, Yang M-C, Kang X-L, Li Y-X, Hong P-P, Zhao X-F, Vasta G, Wang J-X. Scavenger Receptor B2, a Type III Membrane Pattern Recognition Receptor, Senses LPS and Activates the IMD Pathway in Crustaceans.. Proc. Natl. Acad. Sci. USA 2023;120:e2216574120.
doi: 10.1073/pnas.2216574120pmc: PMC10268257pubmed: 37276415google scholar: lookup
de Oliveira FFM, Mamillapalli S, Gonti S, Brey RN, Li H, Schiffer J, Casadevall A, Bann JG. Binding of the von Willebrand Factor a Domain of Capillary Morphogenesis Protein 2 to Anthrax Protective Antigen Vaccine Reduces Immunogenicity in Mice.. mSphere 2020;5:e00556-19.
doi: 10.1128/msphere.00556-19pmc: PMC6968648pubmed: 31941807google scholar: lookup
Sheoran AS, Sponseller BT, Holmes MJ, Timoney JF. Serum and Mucosal Antibody Isotype Responses to M-like Protein (SeM) of Streptococcus equi in Convalescent and Vaccinated Horses.. Vet. Immunol. Immunopathol. 1997;59:239–251.
doi: 10.1016/s0165-2427(97)00074-3pubmed: 9477475google scholar: lookup
Delph KM, Beard LA, Trimble AC, Sutter ME, Timoney JF, Morrow JK. Strangles, Convalescent Streptococcus equi Subspecies equi M Antibody Titers, and Presence of Complications.. J. Vet. Intern. Med. 2018;33:275–279.
doi: 10.1111/jvim.15388pmc: PMC6335513pubmed: 30520521google scholar: lookup
Timoney JF, Eggers D. Serum Bactericidal Responses to Streptococcus equi of Horses Following Infection or Vaccination.. Equine Vet. J. 1985;17:306–310.
doi: 10.1111/j.2042-3306.1985.tb02505.xpubmed: 4076146google scholar: lookup
Newton JR, Verheyen K, Talbot NC, Timoney JF, Wood JLN, Lakhani KH, Chanter N. Control of Strangles Outbreaks by Isolation of Guttural Pouch Carriers Identified Using PCR and Culture of Streptococcus equi.. Equine Vet. J. 2010;32:515–526.
doi: 10.2746/042516400777584721pubmed: 11093626google scholar: lookup
Davidson A, Traub-Dargatz JL, Magnuson RJ, Hill AE, Irwin V, Newton R, Waller A, Smith KC, Callan RJ, Meehan M. Lack of Correlation between Antibody Titers to Fibrinogen-Binding Protein of Streptococcus equi and Persistent Carriers of Strangles.. J. Vet. Diagn. Investig. 2008;20:457–462.
doi: 10.1177/104063870802000407pubmed: 18599850google scholar: lookup