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Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines in porcine plasma.
Vet Immunol Immunopathol. 2013 Jan 15; 151(1-2):28-36.VI

Abstract

Lipopolysaccharide (LPS) has been widely used as a model of immune challenge in pigs as it induces the immediate synthesis of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, which trigger the production of the acute phase proteins (APPs) C-reactive protein (CRP), haptoglobin (Hp) and pig-Major Acute Phase Protein (pig-MAP). To measure secreted proteins in porcine plasma, specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single parameter analysis, yet this approach is time-consuming and expensive for multi-parameter analyses. During the last decade, multiplex bead-based flow cytometry has been increasingly applied as it offers the opportunity to estimate protein ratios in a small sample volume. Cytometric bead array (CBA) is a flow cytometric application using a diversity of beads with unique fluorescence intensities, covalently coupled to a capture antibody for each protein of interest. Detection antibodies, either directly or indirectly conjugated to a fluorochrome, are added to accomplish the desired sandwich format. The aim of the present study was to develop a CBA 3-plex assay for the major pro-inflammatory cytokines TNF-α, IL-1β and IL-6, and an additional CBA 2-plex assay for the major APPs, CRP and pig-MAP, in porcine plasma. Results were compared to commercial ELISA kits. For the CBA 3-plex assay, the limits of detection (LODs) varied between 0.005 and 0.363 ng/mL, the intra- and inter-assay coefficients of variation were <10% and <16%, respectively. For TNF-α, IL-1β, IL-6 and pig-MAP, CBA time-concentration profiles similar to those obtained with commercial ELISAs were observed. In conclusion, the novel validated CBA 3-plex assay provides a fast and economical screening tool for determination of pro-inflammatory cytokine profiles in limited porcine plasma volumes. This tool will be applied to study the immunomodulatory properties of drugs in a porcine LPS inflammation model. This study also demonstrated the applicability of CBA for measurement of APPs in pigs, although a different combination than pig-MAP with CRP is recommended.

Authors+Show Affiliations

Department of Pharmacology, Toxicology and Biochemistry, Ghent University, Faculty of Veterinary Medicine, Salisburylaan 133, 9820 Merelbeke, Belgium. Heidi.Wyns@UGent.beNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Evaluation Study
Journal Article
Validation Study

Language

eng

PubMed ID

23159236

Citation

Wyns, Heidi, et al. "Development of a Cytometric Bead Array Screening Tool for the Simultaneous Detection of Pro-inflammatory Cytokines in Porcine Plasma." Veterinary Immunology and Immunopathology, vol. 151, no. 1-2, 2013, pp. 28-36.
Wyns H, Croubels S, Demeyere K, et al. Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines in porcine plasma. Vet Immunol Immunopathol. 2013;151(1-2):28-36.
Wyns, H., Croubels, S., Demeyere, K., Watteyn, A., De Backer, P., & Meyer, E. (2013). Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines in porcine plasma. Veterinary Immunology and Immunopathology, 151(1-2), 28-36. https://doi.org/10.1016/j.vetimm.2012.09.041
Wyns H, et al. Development of a Cytometric Bead Array Screening Tool for the Simultaneous Detection of Pro-inflammatory Cytokines in Porcine Plasma. Vet Immunol Immunopathol. 2013 Jan 15;151(1-2):28-36. PubMed PMID: 23159236.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines in porcine plasma. AU - Wyns,Heidi, AU - Croubels,Siska, AU - Demeyere,Kristel, AU - Watteyn,Anneleen, AU - De Backer,Patrick, AU - Meyer,Evelyne, Y1 - 2012/10/24/ PY - 2012/05/10/received PY - 2012/09/17/revised PY - 2012/09/28/accepted PY - 2012/11/20/entrez PY - 2012/11/20/pubmed PY - 2013/6/7/medline SP - 28 EP - 36 JF - Veterinary immunology and immunopathology JO - Vet. Immunol. Immunopathol. VL - 151 IS - 1-2 N2 - Lipopolysaccharide (LPS) has been widely used as a model of immune challenge in pigs as it induces the immediate synthesis of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, which trigger the production of the acute phase proteins (APPs) C-reactive protein (CRP), haptoglobin (Hp) and pig-Major Acute Phase Protein (pig-MAP). To measure secreted proteins in porcine plasma, specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single parameter analysis, yet this approach is time-consuming and expensive for multi-parameter analyses. During the last decade, multiplex bead-based flow cytometry has been increasingly applied as it offers the opportunity to estimate protein ratios in a small sample volume. Cytometric bead array (CBA) is a flow cytometric application using a diversity of beads with unique fluorescence intensities, covalently coupled to a capture antibody for each protein of interest. Detection antibodies, either directly or indirectly conjugated to a fluorochrome, are added to accomplish the desired sandwich format. The aim of the present study was to develop a CBA 3-plex assay for the major pro-inflammatory cytokines TNF-α, IL-1β and IL-6, and an additional CBA 2-plex assay for the major APPs, CRP and pig-MAP, in porcine plasma. Results were compared to commercial ELISA kits. For the CBA 3-plex assay, the limits of detection (LODs) varied between 0.005 and 0.363 ng/mL, the intra- and inter-assay coefficients of variation were <10% and <16%, respectively. For TNF-α, IL-1β, IL-6 and pig-MAP, CBA time-concentration profiles similar to those obtained with commercial ELISAs were observed. In conclusion, the novel validated CBA 3-plex assay provides a fast and economical screening tool for determination of pro-inflammatory cytokine profiles in limited porcine plasma volumes. This tool will be applied to study the immunomodulatory properties of drugs in a porcine LPS inflammation model. This study also demonstrated the applicability of CBA for measurement of APPs in pigs, although a different combination than pig-MAP with CRP is recommended. SN - 1873-2534 UR - https://www.unboundmedicine.com/medline/citation/23159236/Development_of_a_cytometric_bead_array_screening_tool_for_the_simultaneous_detection_of_pro_inflammatory_cytokines_in_porcine_plasma_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0165-2427(12)00381-9 DB - PRIME DP - Unbound Medicine ER -