title: Development of a bead-based multiplex assay for the simultaneous detection of porcine inflammation markers using xMAP technology.
abstract: Commercially available assays for the simultaneous detection of multiple inflammatory and cardiac markers in porcine blood samples are currently lacking. Therefore, this study was aimed at developing a bead-based, multiplexed flow cytometric assay to simultaneously detect porcine cytokines [interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor alpha], chemokines (IL-8 and monocyte chemotactic protein 1), growth factors [basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and platelet-derived growth factor-bb], and injury markers (cardiac troponin-I) as well as complement activation markers (C5a and sC5b-9). The method was based on the Luminex xMAP technology, resulting in the assembly of a 6- and 11-plex from the respective individual singleplex situation. The assay was evaluated for dynamic range, sensitivity, cross-reactivity, intra-assay and interassay variance, spike recovery, and correlation between multiplex and commercially available enzyme-linked immunosorbent assay as well as the respective singleplex. The limit of detection ranged from 2.5 to 30,000 pg/ml for all analytes (6- and 11-plex assays), except for soluble C5b-9 with a detection range of 2-10,000 ng/ml (11-plex). Typically, very low cross-reactivity (<3% and <1.4% by 11- and 6-plex, respectively) between analytes was found. Intra-assay variances ranged from 4.9 to 7.4% (6-plex) and 5.3 to 12.9% (11-plex). Interassay variances for cytokines were between 8.1 and 28.8% (6-plex) and 10.1 and 26.4% (11-plex). Correlation coefficients with singleplex assays for 6-plex as well as for 11-plex were high, ranging from 0.988 to 0.997 and 0.913 to 0.999, respectively. In this study, a bead-based porcine 11-plex and 6-plex assay with a good assay sensitivity, broad dynamic range, and low intra-assay variance and cross-reactivity was established. These assays therefore represent a new, useful tool for the analysis of samples generated from experiments with pigs.
authors.affiliation: Department of Clinical Research, University of Bern, 3010 Bern, Switzerland.
authors.forename: Anjan K
authors.initials: AK
authors.lastname: Bongoni
authors.forename: Jonas
authors.initials: J
authors.lastname: Lanz
authors.forename: Robert
authors.initials: R
authors.lastname: Rieben
authors.forename: Yara
authors.initials: Y
authors.lastname: Banz
mesh_terms: D000818:Animals; D001774:Blood Chemical Analysis; D016207:Cytokines / Q000097:blood*; D005434:Flow Cytometry / Q000379:methods*; D007118:Immunoassay; D018836:Inflammation Mediators / Q000097:blood*; D057230:Limit of Detection; D008863:Microspheres; D011137:Polystyrenes / Q000737:chemistry; D015203:Reproducibility of Results; D034421:Sus scrofa
chemical_list: D016207:Cytokines; D018836:Inflammation Mediators; D011137:Polystyrenes