Equine Tumor Necrosis Factor Alpha: Cloning and Expression in Escherichia coli, Generation of Monoclonal Antibodies, and Development of a Sensitive Enzyme-Linked Immunosorbent Assay

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 s...