Monoclonal antibodies to equine interferon-α (IFN-α): New tools to neutralize IFN-activity and to detect secreted IFN-α

Abstract

Interferon-α (IFN-α) 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-α production affects multiple downstream events influencing both innate and adaptive immune responses. Here, we describe the expression of an equine rIFN-α/IgG4 fusion protein in mammalian cells. The anti-viral activity of rIFN-α/IgG4 was found to be 70-fold higher than that of a previously described IFN-γ/IgG1 as tested by bioassay. The purified rIFN-α was subsequently used for the generation of six monoclonal antibodies (mAbs) to equine IFN-α. 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-α 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-α and equine leukocyte IFN, respectively. When analyzing samples with a likely dominance of IFN-α 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 sp 2 = 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-α mAbs are valuable tools to detect native IFN-α for further characterization of the early innate immune response and anti-viral immunity in horses.