Development of monoclonal antibodies for quantification of bovine tumor necrosis factor-α

Summary: We generated a monoclonal antibody (mAb) pair specific to bovine tumor necrosis factor-α (TNF-α) by immunizing one mouse with a recombinant fusion protein of bovine TNF-α and equine IL-4 generated in mammalian cells. Two murine IgG1 mAbs specific to bovine TNF-α were produced in hybridoma cell lines and selected based on their specificity to the recombinant IL-4/TNF-α protein and the native protein produced by peripheral blood mononuclear cells (PBMC). Both mAbs lacked cross-reactivity to equine IL-4 and 3 other recombinant bovine cytokines. A bead-based assay was developed and used to quantify native bovine TNF-α in PBMC culture supernatants and in plasma from ex vivo whole-blood stimulations. In a flow cytometric assay, both mAbs detected intracellular TNF-α in bovine CD14+ monocytes and CD4+/CD8+ lymphocytes. Our bovine TNF-α mAb clones specifically and accurately quantified soluble TNF-α in cell culture supernatants and plasma and detected cytoplasmatic TNF-α in bovine leukocytes.