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Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant

The quantification of cytokines can improve our understanding of immune response and inflammation dynamics in dairy cows. Bead-based assays provide a sensitive, high-throughput platform, allowing for simultaneous quantification of multiple cytokines within a wide linear detection range. Our objectiv...

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Autores principales: Sipka, Anja, Mann, Sabine, Babasyan, Susanna, Freer, Heather, Wagner, Bettina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9623719/
https://www.ncbi.nlm.nih.gov/pubmed/36338808
http://dx.doi.org/10.3168/jdsc.2021-0191
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author Sipka, Anja
Mann, Sabine
Babasyan, Susanna
Freer, Heather
Wagner, Bettina
author_facet Sipka, Anja
Mann, Sabine
Babasyan, Susanna
Freer, Heather
Wagner, Bettina
author_sort Sipka, Anja
collection PubMed
description The quantification of cytokines can improve our understanding of immune response and inflammation dynamics in dairy cows. Bead-based assays provide a sensitive, high-throughput platform, allowing for simultaneous quantification of multiple cytokines within a wide linear detection range. Our objective was to develop a multiplex bead-based assay using monoclonal antibodies for simultaneous quantification of bovine tumor necrosis factor (TNF)-α, IL-10, and IFN-γ in plasma and peripheral blood mononuclear cell (PBMC) culture supernatants. Recombinant cytokine standards produced in mammalian cells were used to determine the lower limit of detection and the linear detection range for each cytokine. The lower limit of detection was 110 pg/mL for IL-10, 95 pg/mL for TNF-α, and 20 pg/mL for IFN-γ. The linear quantification range was 110 to 241,000 pg/mL for IL-10, 95 to 620,000 pg/mL for TNF-α, and 20 to 130,000 pg/mL for IFN-γ. All 3 monoclonal capture and detection antibodies were specific for their respective cytokine analyte when using the recombinant IL-10, TNF-α, and IFN-γ standards. Intraassay and interassay coefficients of variation (CV) were <10% and <12%, respectively, for all analytes and samples matrices. Next, concentrations of native cytokines were determined in PBMC culture supernatants (n = 4) and in plasma from whole-blood samples (n = 6) with or without stimulation with Escherichia coli lipopolysaccharide or a mix of phorbol myristate acetate (PMA) and ionomycin. Peak concentrations of all 3 cytokines were secreted from PBMC after PMA/ionomycin stimulation (TNF-α, 8 h, range: 39,266–506,422 pg/mL; IL-10, 18 h, range: 15,770–63,415 pg/mL; IFN-γ 18 h, range: 189,977–492,659 pg/mL). In contrast, the highest concentrations in plasma from whole-blood stimulation were observed for IL-10 and TNF-α after LPS stimulation (TNF-α, 4 h, range: 1,764–13,460 pg/mL; IL-10, 24 h, range: 2,401–6,371 pg/mL), whereas PMA and ionomycin induced the highest secretion of IFN-γ (18 h, range: 53–20,215 pg/mL). In conclusion, the multiplex assay can quantify native IL-10, TNF-α, and IFN-γ across a broad concentration range in bovine plasma and cell culture supernatant, thereby providing a novel tool to evaluate inflammatory profiles in cattle and especially in dairy cows with inflammatory conditions. The existing multiplex assay can be expanded in the future by adding bead assays for additional bovine cytokines.
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spelling pubmed-96237192022-11-04 Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant Sipka, Anja Mann, Sabine Babasyan, Susanna Freer, Heather Wagner, Bettina JDS Commun Health, Behavior, and Well-being The quantification of cytokines can improve our understanding of immune response and inflammation dynamics in dairy cows. Bead-based assays provide a sensitive, high-throughput platform, allowing for simultaneous quantification of multiple cytokines within a wide linear detection range. Our objective was to develop a multiplex bead-based assay using monoclonal antibodies for simultaneous quantification of bovine tumor necrosis factor (TNF)-α, IL-10, and IFN-γ in plasma and peripheral blood mononuclear cell (PBMC) culture supernatants. Recombinant cytokine standards produced in mammalian cells were used to determine the lower limit of detection and the linear detection range for each cytokine. The lower limit of detection was 110 pg/mL for IL-10, 95 pg/mL for TNF-α, and 20 pg/mL for IFN-γ. The linear quantification range was 110 to 241,000 pg/mL for IL-10, 95 to 620,000 pg/mL for TNF-α, and 20 to 130,000 pg/mL for IFN-γ. All 3 monoclonal capture and detection antibodies were specific for their respective cytokine analyte when using the recombinant IL-10, TNF-α, and IFN-γ standards. Intraassay and interassay coefficients of variation (CV) were <10% and <12%, respectively, for all analytes and samples matrices. Next, concentrations of native cytokines were determined in PBMC culture supernatants (n = 4) and in plasma from whole-blood samples (n = 6) with or without stimulation with Escherichia coli lipopolysaccharide or a mix of phorbol myristate acetate (PMA) and ionomycin. Peak concentrations of all 3 cytokines were secreted from PBMC after PMA/ionomycin stimulation (TNF-α, 8 h, range: 39,266–506,422 pg/mL; IL-10, 18 h, range: 15,770–63,415 pg/mL; IFN-γ 18 h, range: 189,977–492,659 pg/mL). In contrast, the highest concentrations in plasma from whole-blood stimulation were observed for IL-10 and TNF-α after LPS stimulation (TNF-α, 4 h, range: 1,764–13,460 pg/mL; IL-10, 24 h, range: 2,401–6,371 pg/mL), whereas PMA and ionomycin induced the highest secretion of IFN-γ (18 h, range: 53–20,215 pg/mL). In conclusion, the multiplex assay can quantify native IL-10, TNF-α, and IFN-γ across a broad concentration range in bovine plasma and cell culture supernatant, thereby providing a novel tool to evaluate inflammatory profiles in cattle and especially in dairy cows with inflammatory conditions. The existing multiplex assay can be expanded in the future by adding bead assays for additional bovine cytokines. Elsevier 2022-03-03 /pmc/articles/PMC9623719/ /pubmed/36338808 http://dx.doi.org/10.3168/jdsc.2021-0191 Text en © 2022. https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Health, Behavior, and Well-being
Sipka, Anja
Mann, Sabine
Babasyan, Susanna
Freer, Heather
Wagner, Bettina
Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant
title Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant
title_full Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant
title_fullStr Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant
title_full_unstemmed Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant
title_short Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant
title_sort development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant
topic Health, Behavior, and Well-being
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9623719/
https://www.ncbi.nlm.nih.gov/pubmed/36338808
http://dx.doi.org/10.3168/jdsc.2021-0191
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