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

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^® 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-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 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 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.