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 (rETNFalpha), generation and characterization of murine monoclonal antibodies (Mabs) and rabbit polyclonal antibodies (Pabs) against ETNFalpha, and development of a sensitive enzyme-linked immunosorbent assay (ELISA). Genomic-derived DNA sequences encoding mature ETNFalpha were reconstructed by the polymerase chain reaction (PCR) and oligonucleotide-directed mutagenesis and were cloned into the vector pFLAG-1 for expression in Escherichia coli. rETNFalpha 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 rETNFalpha on western blots. Mabs 6H4 and 9B10 recognized similar epitopes on rENTFalpha and neutralized both rETNFalpha and native ETNFalpha (nETNFalpha) 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 ENTFalpha as low as 100 pg/ml and was used to demonstrate the induction of ETNFalpha in horses with experimental endotoxemia. The rETNFalpha, antibodies, and ELISA developed in this report should be useful tools for studies of TNF-mediated diseases in horses.