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 Cardiovascular 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 and Banz Wälti, Yara

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:

01 Jul 2014 15:50

Publisher DOI:

10.1002/cyto.a.22287

PubMed ID:

23576245

URI:

https://boris.unibe.ch/id/eprint/46033

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