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

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Graphical Abstract

Summary: Bead-based assays provide a high-throughput platform allowing for simultaneous quantification of multiple cytokines. Our objective was to develop a multiplex bead-based assay using monoclonal reagents 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 linear detection range for each cytokine. Concentrations of native cytokines were determined in PBMC culture supernatants (n = 4) and plasma from whole-blood samples (n = 6) with or without stimulation with LPS or a mix of phorbol myristate acetate (PMA) and ionomycin. The multiplex assay can quantify native IL-10, TNF-α, and IFN-γ across a broad concentration range in bovine plasma and cell culture samples, thereby providing a novel tool to evaluate inflammatory profiles in cattle, especially in dairy cows with inflammatory conditions.

Highlights

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Bead-based assays provide a platform for simultaneous quantification of multiple cytokines.
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We developed a multiplex bead-based assay for quantification of bovine TNF-α, IL-10, and IFN-γ to evaluate inflammatory profiles in cattle.
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We used mononuclear reagents and recombinant standards produced in mammalian cells.
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The multiplex assay quantified all 3 cytokines across a broad concentration range in plasma and cell culture supernatants.

Abstract

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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Most cited references 27

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LPS-induced cytokine production in human monocytes and macrophages.

Dagmar Quandt, Manuela Rossol, Undine Meusch … (2010)

Lipopolysaccharide (LPS) from Gram-negative bacteria is one of the most potent innate immune-activating stimuli known. Here we review the current understanding of LPS effects on human monocyte and macrophage function. We provide an overview of LPS signal transduction with attention given to receptor cooperativity and species differences in LPS responses, as well as the role of tyrosine phosphorylation and lysine acetylation in signalling. We also review LPS-regulated transcription, with emphasis on chromatin remodeling and primary versus secondary transcriptional control mechanisms. Finally, we review the regulation and function of LPS-inducible cytokines produced by human monocytes and macrophages including TNFα, the IL-1 family, IL-6, IL-8, the IL-10 family, the IL-12 family, IL-15 and TGFβ.

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The inflammasome and danger associated molecular patterns (DAMPs) are implicated in cytokine and chemokine responses following stressor exposure.

Lida Beninson, Kate Tannura, Benjamin N. Greenwood … (2013)

Exposure to stressors or trauma in the absence of pathogenic challenge can stimulate a systemic sterile inflammatory response characterized by high concentrations of blood and tissue cytokines, chemokines, and danger associated molecular patterns (DAMPs) such as heat shock protein-72 (Hsp72), and uric acid. The signaling pathways responsible for these responses remain unclear, however, the inflammasome may play a role. In vitro, DAMPs are known to stimulate the inflammasome in the presence of LPS to activate caspase-1 which cleaves immature precursors of interleukin (IL)-1β and IL-18 into their mature releasable forms. Furthermore, in vivo neutralization of the LPS selectively attenuates the stress-induced increase in the inflammasome-dependent cytokines IL-1β and IL-18. Thus, the current experiments tested the hypothesis that inflammasome-mediated processes are necessary for a systemic stress-induced inflammatory response to an acute stressor. The data presented (1) establish that male F344 rats exposed to an acute severe stressor (100 tail shocks) have elevated plasma concentrations of inflammatory proteins (IL-1β, IL-18, IL-6, IL-10, and monocyte chemotactic protein (MCP)-1), and DAMPs (uric acid and Hsp72); (2) demonstrate that inhibiting caspase-1 in vivo, using the caspase-1 inhibitor ac-YVAD-cmk, attenuates stress-induced production of IL-1β, IL-18, and IL-6 in both the circulation and peripheral tissues; and (3) implicates the DAMPs uric acid and Hsp72 as important signals contributing to inflammasome-dependent inflammatory responses using a stepwise multiple regression. The results increase our mechanistic understanding of systemic sterile inflammatory responses, and provide novel evidence that the inflammasome may be an important pharmacological target for treatment of these conditions. Copyright © 2012 Elsevier Inc. All rights reserved.