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

The expression of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is associated with production losses in dairy cows and is a hallmark of early inflammatory processes. Reliable tools for the detection and quantification of soluble as well as cytoplasmatic bovine TNF-alpha are needed to deepen our understanding of inflammatory dynamics in dairy cows. The objective of this study was to generate a monoclonal antibody (mAb) pair that could be used to quantify bovine TNF-alpha in cell culture supernatants and plasma and to detect cytoplasmatic TNF-alpha in bovine leukocyte populations. One mouse was immunized with a recombinant fusion protein of bovine TNF-alpha and equine IL-4 generated in Chinese hamster ovary cells. Murine monoclonal antibodies specific to bovine TNF-alpha were produced in hybridoma cell lines and selected based on their specificity to the recombinant IL-4/TNF-alpha protein. Clones 197-1 and 65-2, both murine IgG1 isotypes, detected the bovine TNF-alpha fusion protein as well as the native protein produced by peripheral blood mononuclear cells (PBMC) stimulated with a combination of phorbol myristate acetate and ionomycin. Both mAbs were tested for and lacked cross-reactivity to equine IL-4 and 3 other recombinant bovine cytokines (IFN-gamma, IL-10, and CCL5) and were used to develop a fluorescent bead-based assay. The range of bovine TNF-alpha detection in the assay was 0.2 to 620 ng/mL, and the test was used to quantify native bovine TNF-alpha in cell culture supernatants of stimulated PBMC and in plasma from ex vivo whole-blood stimulations. Sample matrices were spiked with TNF-alpha, with subsequent recovery rates (mean +/- SD) of 89% +/- 9 (n = 3) in culture medium and 94% +/- 12 (n = 3) in heat-inactivated fetal bovine serum. Serial dilutions of plasma and cell culture supernatants from stimulated whole blood or PBMC indicated excellent accuracy for quantification of native TNF-alpha in bovine samples. Both bovine TNF-alpha mAbs also detected intracellular TNF-alpha in bovine CD14(+) monocytes and CD4(+)/CD8(+) lymphocytes. In conclusion, we demonstrated that the mAbs generated provide valuable new tools to quantify native bovine TNF-alpha in a wide concentration range and to characterize intracellular TNF-alpha expression in bovine leukocytes.