Development and validation of a sandwich ELISA for equine IL-1β

Abstract In this study, we developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) using newly produced monoclonal antibodies (mAbs) for detecting horse/donkey IL-1β in cell culture medium and serum samples. The mAbs were generated via the use of a KLH-conjugated peptide and purified equine IL-1β protein as separate immunogens. Notably, the generated mAbs (3G8 and 5G3) demonstrated no cross-reactivity with other major inflammatory mediators, including IL-1α, IL-1Ra, TNF-α, and SAA. The IL-1β assay, which is based on the screened mAbs, exhibits a detection range of 200–10,000 pg/mL, meeting clinical detection requirements. The coefficients of variation for the repeatability and reproducibility of the assay were both less than 5%, indicating an acceptable level of variation. Subsequently, 84 equine and 24 asinine serum samples were collected, and the IL-1β concentration was measured with both our assay and a commercial kit in parallel. Our results revealed no significant difference between the in-house and commercial ELISA kits for the detection of IL-1β concentrations in horse sera. Moreover, our ELISA method demonstrated superior sensitivity for IL-1β detection in donkey samples compared to existing commercial assays. These findings suggest that the newly developed ELISA provides a reliable analytical method for detecting IL-1β in both equine and asinine samples.