Production and characterization of two monoclonal antibodies to bovine tumour necrosis factor alpha (TNF-alpha) and their cross-reactivity with ovine TNF-alpha

Tumour necrosis factor alpha (TNF-?) is an innate pro-inflammatory cytokine involved in protection against intracellular pathogens. Existing methods for measuring TNF-? production and function in ruminants are limited to ELISA and many rely on polyclonal antisera. With a view to developing improved detection methods for bovine (bov) TNF-?, monoclonal antibodies (mAb) were produced by immunising mice with a plasmid encoding bov TNF-?. Two of the resulting mAb, termed CC327 and CC328, were used to develop a sandwich ELISA capable of detecting both native and recombinant bov TNF-?. This ELISA did not detect recombinant ovine (ov) TNF-?. A luminometric method was applied to the ELISA to improve sensitivity for detection of native bov TNF-? in culture supernatants derived from bovine monocyte-derived dendritic cells (DC) infected with Mycobacterium bovis. Both CC327 and CC328 detected intracytoplasmic expression of TNF-? in mitogen-activated bovine T lymphocytes. However, only CC328 detected intracytoplasmic ovine TNF-? in transfected cells, explaining the failure of the sandwich ELISA to detect recombinant ov TNF-?. These mAbs have generated the capability to study the role of TNF-? in host immune protection and disease pathogenesis in ruminants.