title: Monoclonal antibodies to equine interferon-alpha (IFN-alpha): new tools to neutralize IFN-activity and to detect secreted IFN-alpha.
abstract: Interferon-alpha (IFN-alpha) is a type I interferon that is secreted during the early stages of the innate immune response and is often induced upon infection with viral pathogens. IFN-alpha production affects multiple downstream events influencing both innate and adaptive immune responses. Here, we describe the expression of an equine rIFN-alpha/IgG4 fusion protein in mammalian cells. The anti-viral activity of rIFN-alpha/IgG4 was found to be 70-fold higher than that of a previously described IFN-gamma/IgG1 as tested by bioassay. The purified rIFN-alpha was subsequently used for the generation of six monoclonal antibodies (mAbs) to equine IFN-alpha. Four of these mAbs inhibited the protective anti-viral effect of equine leukocyte IFN in bioassays. One mAb (clone 240-2) showed a high-neutralizing capacity. An ELISA was established using two anti-equine IFN-alpha mAbs (clones 29B and 240-2) and its analytical sensitivity for was found to be around 800 pg/ml and 3 U/ml for rIFN-alpha and equine leukocyte IFN, respectively. When analyzing samples with a likely dominance of IFN-alpha among type I IFNs, such as supernatants from equine peripheral blood mononuclear cells stimulated with CpG-oligodeoxyribonucleotides, the results obtained by ELISA and IFN bioassay showed a high agreement (r(2)(sp)=0.98). When analyzing samples likely containing a mixture of type I IFNs, such as serum and nasal secretions from virally infected horses, the ELISA only detected some of the IFN-activity recorded in the bioassay. Overall, the data showed that the new anti-equine IFN-alpha mAbs are valuable tools to detect native IFN-alpha for further characterization of the early innate immune response and anti-viral immunity in horses.
authors.affiliation: Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. bw73@cornell.edu
authors.forename: Bettina
authors.initials: B
authors.lastname: Wagner
authors.forename: Julie M
authors.initials: JM
authors.lastname: Hillegas
authors.forename: M Julia B F
authors.initials: MJ
authors.lastname: Flaminio
authors.forename: Eva
authors.initials: E
authors.lastname: Wattrang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D001681:Biological Assay; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D006736:Horses; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D016898:Interferon-alpha / Q000032:analysis / Q000037:antagonists & inhibitors / Q000276:immunology*; D007962:Leukocytes / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins