Optimization of a bovine cytokine multiplex assay using a new bovine and cross-reactive equine monoclonal antibodies

Sipka, Anja; Babasyan, Susanna; Asbie, Sanda; Freer, Heather; Wagner, Bettina

doi:10.1016/j.vetimm.2024.110789

Cited by 2 publications

(3 citation statements)

References 34 publications

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“…The multiplex assay was performed, as previously described ( 43 ). All antibodies used are monoclonal antibodies (mAbs) of IgG1 isotypes and were generated in mice.…”

Section: Methodsmentioning

confidence: 99%

“…All antibodies used are monoclonal antibodies (mAbs) of IgG1 isotypes and were generated in mice. Equine antibodies used have been shown to be cross-reactive with bovine proteins ( 43 ). If not indicated otherwise, mAbs were produced in house ( 43 – 46 ).…”

Section: Methodsmentioning

confidence: 99%

“…Equine antibodies used have been shown to be cross-reactive with bovine proteins ( 43 ). If not indicated otherwise, mAbs were produced in house ( 43 – 46 ). Briefly, fluorescent beads were coupled with bovine interleukin-10 (IL10) mAb 130, equine interferon gamma (IFNγ) mAb 3, and tumor necrosis factor alpha (TNFα) mAb 197–1.…”

Section: Methodsmentioning

confidence: 99%

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The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

Harman,

Sipka,

Oxford

et al. 2024

Front. Immunol.

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BackgroundInnovative therapies against bacterial infections are needed. One approach is to focus on host-directed immunotherapy (HDT), with treatments that exploit natural processes of the host immune system. The goals of this type of therapy are to stimulate protective immunity while minimizing inflammation-induced tissue damage. We use non-traditional large animal models to explore the potential of the mammosphere-derived epithelial cell (MDEC) secretome, consisting of all bioactive factors released by the cells, to modulate host immune functions. MDEC cultures are enriched for mammary stem and progenitor cells and can be generated from virtually any mammal. We previously demonstrated that the bovine MDEC secretome, collected and delivered as conditioned medium (CM), inhibits the growth of bacteria in vitro and stimulates functions related to tissue repair in cultured endothelial and epithelial cells.MethodsThe immunomodulatory effects of the bovine MDEC secretome on bovine neutrophils, an innate immune cell type critical for resolving bacterial infections, were determined in vitro using functional assays. The effects of MDEC CM on neutrophil molecular pathways were explored by evaluating the production of specific cytokines by neutrophils and examining global gene expression patterns in MDEC CM-treated neutrophils. Enzyme linked immunosorbent assays were used to determine the concentrations of select proteins in MDEC CM and siRNAs were used to reduce the expression of specific MDEC-secreted proteins, allowing for the identification of bioactive factors modulating neutrophil functions.ResultsNeutrophils exposed to MDEC secretome exhibited increased chemotaxis and phagocytosis and decreased intracellular reactive oxygen species and extracellular trap formation, when compared to neutrophils exposed to control medium. C-X-C motif chemokine 6, superoxide dismutase, peroxiredoxin-2, and catalase, each present in the bovine MDEC secretome, were found to modulate neutrophil functions.ConclusionThe MDEC secretome administered to treat bacterial infections may increase neutrophil recruitment to the site of infection, stimulate pathogen phagocytosis by neutrophils, and reduce neutrophil-produced ROS accumulation. As a result, pathogen clearance might be improved and local inflammation and tissue damage reduced.

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“…The multiplex assay was performed, as previously described ( 43 ). All antibodies used are monoclonal antibodies (mAbs) of IgG1 isotypes and were generated in mice.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

Harman,

Sipka,

Oxford

et al. 2024

Front. Immunol.

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Development of a monoclonal antibody against duck IFN-γ protein and the application for Intracellular Cytokine Staining

Chen,

Song,

Chen

et al. 2024

Preprint

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Interferon-γ (IFN-γ), a member of the type II IFN family, is a crucial cytokine in the immune system and serves as an important indicator of immune response. Intracellular Cytokine Staining (ICS) is a technique used to analyze the production of cytokines within individual cells, and it has a wide range of applications in the fields of immunological monitoring, vaccine trials, and the study of infectious diseases. This study aimed to prepare monoclonal antibodies against duck IFN-γ protein and to establish an ICS protocol for detecting the duck IFN-γ protein. The duIFN-γ-His or duIFN-γ-Fc gene was cloned into the pEE12.4 expression vector and expressed as a recombinant protein of size 20.2 KDa or 54.9 KDa in 293F cells. The purified recombinant proteins were inoculated into BALB/c mice to generate splenic lymphocytes capable of secreting anti-duIFN-γ antibodies, and hybridoma cells were obtained after fusion with SP2/0 cells. A new hybridoma cell line named 24H4, which stably secreted IgG3 κ subtype antibody against duck IFN-γ. This monoclonal antibody (mAb) was identified by Western Blot to recognize duck IFN-γ antibodies, and the indirect ELISA results showed that its ability to recognize IFN-γ protein reached 0.001 μg/ml. The established ICS method was used to stain PBMCs after ConA stimulation, and duck IFN-γ protein was successfully detected by flow cytometry, indicating that the ICS method was successfully established. This study we use the monoclonal antibody, 24H4, provides a crucial tool for subsequent research on duck cellular immune responses.

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Optimization of a bovine cytokine multiplex assay using a new bovine and cross-reactive equine monoclonal antibodies

Cited by 2 publications

References 34 publications

The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

Development of a monoclonal antibody against duck IFN-γ protein and the application for Intracellular Cytokine Staining

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