Development of monoclonal antibodies for quantification of bovine tumor necrosis factor-α
The expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) is associated with production losses in dairy cows and is a hallmark of early inflammatory processes. Reliable tools for the detection and quantification of soluble as well as cytoplasmatic bovine TNF-α are needed to deep...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-11-01
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| Series: | JDS Communications |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666910221001617 |
| _version_ | 1797788469011939328 |
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| author | Anja Sipka Susanna Babasyan Sabine Mann Heather Freer Suzanne Klaessig Bettina Wagner |
| author_facet | Anja Sipka Susanna Babasyan Sabine Mann Heather Freer Suzanne Klaessig Bettina Wagner |
| author_sort | Anja Sipka |
| collection | DOAJ |
| description | The expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) is associated with production losses in dairy cows and is a hallmark of early inflammatory processes. Reliable tools for the detection and quantification of soluble as well as cytoplasmatic bovine TNF-α are needed to deepen our understanding of inflammatory dynamics in dairy cows. The objective of this study was to generate a monoclonal antibody (mAb) pair that could be used to quantify bovine TNF-α in cell culture supernatants and plasma and to detect cytoplasmatic TNF-α in bovine leukocyte populations. One mouse was immunized with a recombinant fusion protein of bovine TNF-α and equine IL-4 generated in Chinese hamster ovary cells. Murine monoclonal antibodies specific to bovine TNF-α were produced in hybridoma cell lines and selected based on their specificity to the recombinant IL-4/TNF-α protein. Clones 197-1 and 65-2, both murine IgG1 isotypes, detected the bovine TNF-α fusion protein as well as the native protein produced by peripheral blood mononuclear cells (PBMC) stimulated with a combination of phorbol myristate acetate and ionomycin. Both mAbs were tested for and lacked cross-reactivity to equine IL-4 and 3 other recombinant bovine cytokines (IFN-γ, IL-10, and CCL5) and were used to develop a fluorescent bead-based assay. The range of bovine TNF-α detection in the assay was 0.2 to 620 ng/mL, and the test was used to quantify native bovine TNF-α in cell culture supernatants of stimulated PBMC and in plasma from ex vivo whole-blood stimulations. Sample matrices were spiked with TNF-α, with subsequent recovery rates (mean ± SD) of 89% ± 9 (n = 3) in culture medium and 94% ± 12 (n = 3) in heat-inactivated fetal bovine serum. Serial dilutions of plasma and cell culture supernatants from stimulated whole blood or PBMC indicated excellent accuracy for quantification of native TNF-α in bovine samples. Both bovine TNF-α mAbs also detected intracellular TNF-α in bovine CD14+ monocytes and CD4+/CD8+ lymphocytes. In conclusion, we demonstrated that the mAbs generated provide valuable new tools to quantify native bovine TNF-α in a wide concentration range and to characterize intracellular TNF-α expression in bovine leukocytes. |
| first_indexed | 2024-03-13T01:35:59Z |
| format | Article |
| id | doaj.art-8d4c6ec185f446c696612b5a9b0dab73 |
| institution | Directory Open Access Journal |
| issn | 2666-9102 |
| language | English |
| last_indexed | 2024-03-13T01:35:59Z |
| publishDate | 2021-11-01 |
| publisher | Elsevier |
| record_format | Article |
| series | JDS Communications |
| spelling | doaj.art-8d4c6ec185f446c696612b5a9b0dab732023-07-04T05:10:44ZengElsevierJDS Communications2666-91022021-11-0126415420Development of monoclonal antibodies for quantification of bovine tumor necrosis factor-αAnja Sipka0Susanna Babasyan1Sabine Mann2Heather Freer3Suzanne Klaessig4Bettina Wagner5Corresponding author; Department for Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY 14853Department for Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY 14853Department for Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY 14853Department for Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY 14853Department for Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY 14853Department for Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY 14853The expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) is associated with production losses in dairy cows and is a hallmark of early inflammatory processes. Reliable tools for the detection and quantification of soluble as well as cytoplasmatic bovine TNF-α are needed to deepen our understanding of inflammatory dynamics in dairy cows. The objective of this study was to generate a monoclonal antibody (mAb) pair that could be used to quantify bovine TNF-α in cell culture supernatants and plasma and to detect cytoplasmatic TNF-α in bovine leukocyte populations. One mouse was immunized with a recombinant fusion protein of bovine TNF-α and equine IL-4 generated in Chinese hamster ovary cells. Murine monoclonal antibodies specific to bovine TNF-α were produced in hybridoma cell lines and selected based on their specificity to the recombinant IL-4/TNF-α protein. Clones 197-1 and 65-2, both murine IgG1 isotypes, detected the bovine TNF-α fusion protein as well as the native protein produced by peripheral blood mononuclear cells (PBMC) stimulated with a combination of phorbol myristate acetate and ionomycin. Both mAbs were tested for and lacked cross-reactivity to equine IL-4 and 3 other recombinant bovine cytokines (IFN-γ, IL-10, and CCL5) and were used to develop a fluorescent bead-based assay. The range of bovine TNF-α detection in the assay was 0.2 to 620 ng/mL, and the test was used to quantify native bovine TNF-α in cell culture supernatants of stimulated PBMC and in plasma from ex vivo whole-blood stimulations. Sample matrices were spiked with TNF-α, with subsequent recovery rates (mean ± SD) of 89% ± 9 (n = 3) in culture medium and 94% ± 12 (n = 3) in heat-inactivated fetal bovine serum. Serial dilutions of plasma and cell culture supernatants from stimulated whole blood or PBMC indicated excellent accuracy for quantification of native TNF-α in bovine samples. Both bovine TNF-α mAbs also detected intracellular TNF-α in bovine CD14+ monocytes and CD4+/CD8+ lymphocytes. In conclusion, we demonstrated that the mAbs generated provide valuable new tools to quantify native bovine TNF-α in a wide concentration range and to characterize intracellular TNF-α expression in bovine leukocytes.http://www.sciencedirect.com/science/article/pii/S2666910221001617 |
| spellingShingle | Anja Sipka Susanna Babasyan Sabine Mann Heather Freer Suzanne Klaessig Bettina Wagner Development of monoclonal antibodies for quantification of bovine tumor necrosis factor-α JDS Communications |
| title | Development of monoclonal antibodies for quantification of bovine tumor necrosis factor-α |
| title_full | Development of monoclonal antibodies for quantification of bovine tumor necrosis factor-α |
| title_fullStr | Development of monoclonal antibodies for quantification of bovine tumor necrosis factor-α |
| title_full_unstemmed | Development of monoclonal antibodies for quantification of bovine tumor necrosis factor-α |
| title_short | Development of monoclonal antibodies for quantification of bovine tumor necrosis factor-α |
| title_sort | development of monoclonal antibodies for quantification of bovine tumor necrosis factor α |
| url | http://www.sciencedirect.com/science/article/pii/S2666910221001617 |
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