Development of a bead-based multiplexed assay for simultaneous quantification of five bovine cytokines by flow cytometry

Rodrigues Valérie, Baudier Jean Baptiste, Chantal Isabelle. 2017. Development of a bead-based multiplexed assay for simultaneous quantification of five bovine cytokines by flow cytometry. Cytometry. Part A, 91 (9), n.spéc. ISAC-Cytometry Part A Mentorship Program Special Section on Imaging Cytometry: Automated Morphology andFeature Extraction : 901-907.

Quartile : Q2, Sujet : BIOCHEMICAL RESEARCH METHODS / Quartile : Q3, Sujet : CELL BIOLOGY

Résumé : Quantifying cytokines is extremely important in studies of host–pathogen interactions. Multiplex assays are commercially available but only for human and mouse cytokines. Here a method for the simultaneous quantification of five important bovine cytokines IFNγ, IL-4, IL-10, IL-12, and TNFα in cell culture supernatants, using flow cytometry was reported. Functional beads from BD Biosciences expressing specific APC intensity were used. Commercially available antibodies against bovine cytokines were covalently coupled to beads as capture antibodies. Fixed recombinant cytokines were revealed with a second monoclonal antibody coupled with biotin, then revealed with streptavidin–PE. This complex was analyzed using a standard flow cytometer. Experiments were performed to check no cross reactions had occurred. The limits of detection ranged between 0.08 and 0.4 ng/ml depending on the cytokine, and the linearity between the lower and higher limits was remarkable (R2 > 99.8%). Finally, native cytokines from cell culture supernatants were tested. Results were compared using the standard ELISA test and showed that concentrations of native cytokine in cell culture supernatants were comparable with the two methods, with a wider dynamic range using beads and flow cytometry than with ELISA assays. Bovine IFNγ, IL-4, IL-10, IL-12, and TNFα in culture supernatants can be now simultaneously detected in a single assay, using a standard flow cytometer for both basic and high-throughput analyses.

Mots-clés Agrovoc : cytométrie en flux (cellules), cytokine, culture de cellule, mode de culture, Test ELISA

Classification Agris : L73 - Maladies des animaux
U30 - Méthodes de recherche

Champ stratégique Cirad : Axe 4 (2014-2018) - Santé des animaux et des plantes

Auteurs et affiliations

• Rodrigues Valérie, CIRAD-BIOS-UMR ASTRE (FRA)
• Baudier Jean Baptiste, INRA (FRA)
• Chantal Isabelle, CIRAD-BIOS-UMR INTERTRYP (FRA) ORCID: 0000-0002-3429-484X

Source : Cirad-Agritrop (https://agritrop.cirad.fr/586415/)

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