Abstract
We describe the production and purification of recombinant equine tumor necrosis factor alpha (rETNFα), generation and characterization of murine monoclonal antibodies (Mabs) and rabbit polyclonal antibodies (Pabs) against ETNFα, and development of a sensitive enzyme-linked immunosorbent assay (ELISA). Genomic-derived DNA sequences encoding mature ETNFα were reconstructed by the polymerase chain reaction (PCR) and oligonucleotidedirected mutagenesis and were cloned into the vector pFLAG-1 for expression in Escherichia coli. rETNFα was purified by anti-FLAG immunoaffinity chromatography and then used as immunogen for production of murine Mabs and rabbit Pabs. Three Mabs (6H4, 9B10, and 12F6) were obtained from one fusion. All three Mabs recognized rETNFα on western blots. Mabs 6H4 and 9B10 recognized similar epitopes on rENTFα and neutralized both rETNFα and native ETNFα (nETNFα) in a WEHI cell cytotoxicity assay. A sensitive ELISA was developed using Mab 6H4 and biotin-labeled rabbit Pabs. The ELISA was shown to detect levels of ENTFα as low as 100 pg/ml and was used to demonstrate the induction of ETNFα in horses with experimental endotoxemia. The rETNFα, antibodies, and ELISA developed in this report should be useful tools for studies of TNF-mediated diseases in horses.
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