title: Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines in porcine plasma.
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.affiliation: Department of Pharmacology, Toxicology and Biochemistry, Ghent University, Faculty of Veterinary Medicine, Salisburylaan 133, 9820 Merelbeke, Belgium. Heidi.Wyns@UGent.be
authors.forename: Heidi
authors.initials: H
authors.lastname: Wyns
authors.forename: Siska
authors.initials: S
authors.lastname: Croubels
authors.forename: Kristel
authors.initials: K
authors.lastname: Demeyere
authors.forename: Anneleen
authors.initials: A
authors.lastname: Watteyn
authors.forename: Patrick
authors.initials: P
authors.lastname: De Backer
authors.forename: Evelyne
authors.initials: E
authors.lastname: Meyer
mesh_terms: D000209:Acute-Phase Proteins / Q000032:analysis; D000818:Animals; D000906:Antibodies; D002097:C-Reactive Protein / Q000032:analysis; D016207:Cytokines / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D007118:Immunoassay / Q000379:methods; D018836:Inflammation Mediators / Q000097:blood*; D053583:Interleukin-1beta / Q000097:blood; D015850:Interleukin-6 / Q000097:blood; D008070:Lipopolysaccharides / Q000276:immunology / Q000494:pharmacology; D008297:Male; D023421:Models, Animal; D018448:Models, Immunological; D034421:Sus scrofa / Q000097:blood* / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000097:blood
chemical_list: D000209:Acute-Phase Proteins; D000906:Antibodies; D002097:C-Reactive Protein; D016207:Cytokines; D005456:Fluorescent Dyes; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; C095815:ITIH4 protein, Sus scrofa; C440499:lipopolysaccharide, Escherichia coli O111 B4