Development of a bead-based multiplex assay for the simultaneous detection of porcine inflammation markers using xMAP technology

Bongoni, Anjan K.; Lanz, Jonas; Rieben, Robert; Banz Wälti, Yara (2013). Development of a bead-based multiplex assay for the simultaneous detection of porcine inflammation markers using xMAP technology. Cytometry Part A, 83A(7), pp. 636-647. Wiley 10.1002/cyto.a.22287

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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.

Item Type:	Journal Article (Original Article)
Division/Institute:	04 Faculty of Medicine > Service Sector > Institute of Pathology > Clinical Pathology 04 Faculty of Medicine > Department of Cardiovascular Disorders (DHGE) > Clinic of Heart Surgery 04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Forschungsbereich Mu50 > Forschungsgruppe Herz und Gefässe 04 Faculty of Medicine > Service Sector > Institute of Pathology 04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR)
UniBE Contributor:	Lanz, Jonas, Rieben, Robert, Banz Wälti, Yara Sarah
Subjects:	600 Technology > 610 Medicine & health 500 Science > 570 Life sciences; biology
ISSN:	1552-4930
Publisher:	Wiley
Language:	English
Submitter:	Verena de Serra Frazao-Bill
Date Deposited:	21 May 2014 11:36
Last Modified:	27 Feb 2024 14:29
Publisher DOI:	10.1002/cyto.a.22287
PubMed ID:	23576245
URI:	https://boris.unibe.ch/id/eprint/46033