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.
Publication Date: 2008-05-24 PubMed ID: 18586327DOI: 10.1016/j.vetimm.2008.05.016Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
Summary
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This research focused on creating and testing monoclonal antibodies for equine interferon-alpha (IFN-alpha), which plays an important role in the early stages of immune responses in horses. These antibodies would allow for more effective detection, characterization, and neutralization of IFN-alpha in horses, and could ultimately enhance understanding of their immunity.
Study Overview
- The research primarily centered on the characterisation and expression of equine rIFN-alpha/IgG4, a fusion protein in mammalian cells. The anti-viral activity levels of this protein were observed to be significantly higher than the previously studied IFN-gamma/IgG1 protein.
- Researchers also produced six monoclonal antibodies (mAbs) that targeted equine IFN-alpha. Monoclonal antibodies are laboratory-made molecules that can precisely bind to certain proteins (in this case IFN-alpha).
Results of The Antiviral Activity Test
- Of the six mAbs created, four had the ability to inhibit the antiviral effect generated by equine leukocyte (a type of white blood cell) IFN, highlighting their potential for neutralizing IFN-activity. And, one mAb, known as clone 240-2, showed a high capacity for neutralization.
Testing Detection and Sensitivity
- Taking two of the anti-equine IFN-alpha mAbs (clones 29B and 240-2), an Enzyme-Linked Immunosorbent Assay (ELISA) was established. This is a test that measures the concentration of antibodies in the blood and was used to determine the sensitivity of these mAbs to rIFN-alpha and equine leukocyte IFN.
- The concentrations required to detect these IFNs were reported as around 800 pg/ml and 3 U/ml, respectively.
Comparative Analysis of ELISA and Bioassay
- The team analyzed supernatants from equine peripheral blood mononuclear cells stimulated with CpG-oligonucleotides. These are particular stimulated cell samples that have a dominance of IFN-alpha among type I IFNs.
- The results from the ELISA tests showed a high agreement with those from IFN bioassays.
- However, it was observed that ELISA tests detected only a portion of the IFN-activity recorded by the bioassay in samples from virally infected horses containing a mixture of type I IFNs.
Conclusion
- The results of the research highlight the potential of the new anti-equine IFN-alpha mAbs to serve as essential tools in identifying native IFN-alpha. This could lead to more detailed knowledge concerning the early innate immune response and anti-viral immunity in horses.
Cite This Article
APA
Wagner B, Hillegas JM, Flaminio MJ, Wattrang E.
(2008).
Monoclonal antibodies to equine interferon-alpha (IFN-alpha): new tools to neutralize IFN-activity and to detect secreted IFN-alpha.
Vet Immunol Immunopathol, 125(3-4), 315-325.
https://doi.org/10.1016/j.vetimm.2008.05.016 Publication
Researcher Affiliations
- Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. bw73@cornell.edu
MeSH Terms
- Animals
- Antibodies, Monoclonal / biosynthesis
- Antibodies, Monoclonal / chemistry
- Antibodies, Monoclonal / immunology
- Antibodies, Monoclonal / pharmacology
- Biological Assay
- CHO Cells
- Cricetinae
- Cricetulus
- Enzyme-Linked Immunosorbent Assay / veterinary
- Horses
- Immunoglobulin G / biosynthesis
- Immunoglobulin G / genetics
- Immunoglobulin G / immunology
- Interferon-alpha / analysis
- Interferon-alpha / antagonists & inhibitors
- Interferon-alpha / immunology
- Leukocytes / immunology
- Recombinant Fusion Proteins / biosynthesis
- Recombinant Fusion Proteins / genetics
- Recombinant Fusion Proteins / immunology
- Recombinant Proteins / biosynthesis
- Recombinant Proteins / genetics
- Recombinant Proteins / immunology
- Transfection
Citations
This article has been cited 5 times.- Wu X, Yang W, Cheng JG, Luo Y, Fu WL, Zhou L, Wu J, Wang Y, Zhong ZJ, Yang ZX, Yao XP, Ren MS, Li YM, Liu J, Ding H, Chen JN. Molecular cloning, prokaryotic expression and its application potential evaluation of interferon (IFN)-ω of forest musk deer. Sci Rep 2023 Jun 30;13(1):10625.
- Sipka A, Babasyan S, Mann S, Freer H, Klaessig S, Wagner B. Development of monoclonal antibodies for quantification of bovine tumor necrosis factor-α. JDS Commun 2021 Nov;2(6):415-420.
- Wimer CL, Schnabel CL, Perkins G, Babasyan S, Freer H, Stout AE, Rollins A, Osterrieder N, Goodman LB, Glaser A, Wagner B. The deletion of the ORF1 and ORF71 genes reduces virulence of the neuropathogenic EHV-1 strain Ab4 without compromising host immunity in horses. PLoS One 2018;13(11):e0206679.
- Schnabel CL, Wimer CL, Perkins G, Babasyan S, Freer H, Watts C, Rollins A, Osterrieder N, Wagner B. Deletion of the ORF2 gene of the neuropathogenic equine herpesvirus type 1 strain Ab4 reduces virulence while maintaining strong immunogenicity. BMC Vet Res 2018 Aug 22;14(1):245.
- Detournay O, Morrison DA, Wagner B, Zarnegar B, Wattrang E. Genomic analysis and mRNA expression of equine type I interferon genes. J Interferon Cytokine Res 2013 Dec;33(12):746-59.
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