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Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine, that is involved in acute inflammation and is employed as a biomarker of inflammatory diseases in several species for which reliable quantification is available. We aimed to develop suitable tools to quantify TNF-α in equine samples. We generated two new mAbs against equine TNF-α (clones 48 and 292), evaluated their specificity for this cytokine, and confirmed detection of native TNF-α in stimulated equine PBMC. The TNF-α mAbs were paired in a fluorescent bead-based assay for qu