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-alpha) is an innate pro-inflammatory cytokine involved in protection against intracellular pathogens. Existing methods for measuring TNF-alpha 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 (boy) TNF-alpha, monoclonal antibodies (mAb) were produced by immunising mice with a plasmid encoding boy TNF-alpha. Two of the resulting mAb, termed CC327 and CC328, were used to develop a sandwich ELISA capable of detecting both native and recombinant boy TNF-alpha. This ELISA did not detect recombinant ovine (ov) TNF-alpha. A luminometric method was applied to the ELISA to improve sensitivity for detection of native boy TNF-alpha in culture supernatants derived from bovine monocyte-derived dendritic cells (DC) infected with Mycobacterium bovis. Both CC327 and CC328 detected intracytoplasmic expression of TNF-alpha in mitogen-activated bovine T lymphocytes. However, only CC328 detected intracytoplasmic ovine TNF-alpha in transfected cells, explaining the failure of the sandwich ELISA to detect recombinant ov TNF-alpha. These mAbs have generated the capability to study the role of TNF-alpha in host immune protection and disease pathogenesis in ruminants. (C) 2010 Elsevier B.V. All rights reserved.