Development and characterization of monoclonal antibodies specific for chicken interleukin-13 and their neutralizing effects in chicken primary monocytes
Compared with mammals, the functionality of chicken cytokines is not well understood because of the unavailability of immune reagents. Mammalian interleukin (IL)-13 is an important Th2 type cytokine with well-known biological functions through its 2 receptors, IL-13 receptor (IL-13R)-α1 and IL-13Rα2...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2020-02-01
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| Series: | Poultry Science |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0032579119441813 |
| _version_ | 1828484505287000064 |
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| author | Atul A. Chaudhari Woo H. Kim Hyun S. Lillehoj |
| author_facet | Atul A. Chaudhari Woo H. Kim Hyun S. Lillehoj |
| author_sort | Atul A. Chaudhari |
| collection | DOAJ |
| description | Compared with mammals, the functionality of chicken cytokines is not well understood because of the unavailability of immune reagents. Mammalian interleukin (IL)-13 is an important Th2 type cytokine with well-known biological functions through its 2 receptors, IL-13 receptor (IL-13R)-α1 and IL-13Rα2. In the present study, we developed mouse monoclonal antibodies (mAb) against chIL-13 and further investigated their specificity in detecting endogenously produced chIL-13. Upon characterization of mAb using indirect ELISA and Western blot, the capture ELISA was developed for detecting chIL-13. Neutralizing effects were tested by measuring nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in primary chicken monocytes stimulated with chIL-13, lipopolysaccharide (LPS), chIL-13+LPS, or chIL-13+LPS+mAb. In addition, gene expression of chIL-13Rα1, chIL-13Rα2, and TGF-β1 was tested in chicken monocytes treated with chIL-13 or chIL-13+mAb. Based on indirect ELISA, 5 mAb that detected recombinant chIL-13 were identified, and all of them specifically detected recombinant chIL-13 protein by Western blotting. An optimal signal was obtained with 2 mAb (#9B11 and #10A2) in a pairing assay, and these 2 mAb were used in a capture assay. A neutralization assay further revealed that chIL-13 reduced LPS-stimulated NO production and iNOS expression in monocytes and macrophage cells, and the 2 mAb (#9B11 and #10A2) abrogated these effects. In addition, chIL-13–induced expressions of chIL-13Rα2 and TGF-β1 were neutralized by the 2 mAb. In summary, the present study showed that chIL-13 may be involved in the alternative activation of primary monocytes in chickens and that chIL-13 signaling may be regulated through chIL-13Rα2 binding and TGF-β1 secretion. Importantly, the newly developed anti–chIL-13 mAb will serve as valuable immune reagents for future studies on the biological activity of chIL-13 and its receptors. |
| first_indexed | 2024-12-11T08:57:01Z |
| format | Article |
| id | doaj.art-17c716c044e64189bb02cd0a0565cc30 |
| institution | Directory Open Access Journal |
| issn | 0032-5791 |
| language | English |
| last_indexed | 2024-12-11T08:57:01Z |
| publishDate | 2020-02-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Poultry Science |
| spelling | doaj.art-17c716c044e64189bb02cd0a0565cc302022-12-22T01:13:52ZengElsevierPoultry Science0032-57912020-02-01992772782Development and characterization of monoclonal antibodies specific for chicken interleukin-13 and their neutralizing effects in chicken primary monocytesAtul A. Chaudhari0Woo H. Kim1Hyun S. Lillehoj2Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, ARS, U.S. Department of Agriculture, Beltsville, MD 20705, USAAnimal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, ARS, U.S. Department of Agriculture, Beltsville, MD 20705, USACorresponding author:; Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, ARS, U.S. Department of Agriculture, Beltsville, MD 20705, USACompared with mammals, the functionality of chicken cytokines is not well understood because of the unavailability of immune reagents. Mammalian interleukin (IL)-13 is an important Th2 type cytokine with well-known biological functions through its 2 receptors, IL-13 receptor (IL-13R)-α1 and IL-13Rα2. In the present study, we developed mouse monoclonal antibodies (mAb) against chIL-13 and further investigated their specificity in detecting endogenously produced chIL-13. Upon characterization of mAb using indirect ELISA and Western blot, the capture ELISA was developed for detecting chIL-13. Neutralizing effects were tested by measuring nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in primary chicken monocytes stimulated with chIL-13, lipopolysaccharide (LPS), chIL-13+LPS, or chIL-13+LPS+mAb. In addition, gene expression of chIL-13Rα1, chIL-13Rα2, and TGF-β1 was tested in chicken monocytes treated with chIL-13 or chIL-13+mAb. Based on indirect ELISA, 5 mAb that detected recombinant chIL-13 were identified, and all of them specifically detected recombinant chIL-13 protein by Western blotting. An optimal signal was obtained with 2 mAb (#9B11 and #10A2) in a pairing assay, and these 2 mAb were used in a capture assay. A neutralization assay further revealed that chIL-13 reduced LPS-stimulated NO production and iNOS expression in monocytes and macrophage cells, and the 2 mAb (#9B11 and #10A2) abrogated these effects. In addition, chIL-13–induced expressions of chIL-13Rα2 and TGF-β1 were neutralized by the 2 mAb. In summary, the present study showed that chIL-13 may be involved in the alternative activation of primary monocytes in chickens and that chIL-13 signaling may be regulated through chIL-13Rα2 binding and TGF-β1 secretion. Importantly, the newly developed anti–chIL-13 mAb will serve as valuable immune reagents for future studies on the biological activity of chIL-13 and its receptors.http://www.sciencedirect.com/science/article/pii/S0032579119441813interleukin-13antigen capture assaymonoclonal antibodieschickenalternative activation |
| spellingShingle | Atul A. Chaudhari Woo H. Kim Hyun S. Lillehoj Development and characterization of monoclonal antibodies specific for chicken interleukin-13 and their neutralizing effects in chicken primary monocytes Poultry Science interleukin-13 antigen capture assay monoclonal antibodies chicken alternative activation |
| title | Development and characterization of monoclonal antibodies specific for chicken interleukin-13 and their neutralizing effects in chicken primary monocytes |
| title_full | Development and characterization of monoclonal antibodies specific for chicken interleukin-13 and their neutralizing effects in chicken primary monocytes |
| title_fullStr | Development and characterization of monoclonal antibodies specific for chicken interleukin-13 and their neutralizing effects in chicken primary monocytes |
| title_full_unstemmed | Development and characterization of monoclonal antibodies specific for chicken interleukin-13 and their neutralizing effects in chicken primary monocytes |
| title_short | Development and characterization of monoclonal antibodies specific for chicken interleukin-13 and their neutralizing effects in chicken primary monocytes |
| title_sort | development and characterization of monoclonal antibodies specific for chicken interleukin 13 and their neutralizing effects in chicken primary monocytes |
| topic | interleukin-13 antigen capture assay monoclonal antibodies chicken alternative activation |
| url | http://www.sciencedirect.com/science/article/pii/S0032579119441813 |
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