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Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines and acute phase proteins in porcine plasma

Heidi Wyns UGent, Siska Croubels UGent, Kristel Demeyere UGent, Anneleen Watteyn UGent, Patrick De Backer UGent and Evelyne Meyer UGent (2012) JOURNAL OF VETERINARY PHARMACOLOGY AND THERAPEUTICS.
abstract
Introduction : Multiplex assays currently are a very popular tool for the simultaneous detection of biomarkers of infection and inflammation. Whereas specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single factor analysis, for multi-parameter analyses, this approach is time-consuming and expensive. Cytometric bead array (CBA) is a flexible, bead-based flow cytometric application for the simultaneous detection of various soluble proteins of interest. Therefore, the aim of the present study was to develop and validate 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 acute phase proteins (APPs) CRP and pig-MAP in plasma of lipopolysaccharide (LPS)-challenged pigs. Furthermore, results were compared to commercial ELISA kits. Materials and Methods : Four pigs with a mean body weight (BW) of 24.9 kg were intravenously challenged with 15 µg ultrapure LPS/kg BW (Escherichia coli serotype O111:B4). Plasma was isolated and stored at -70°C until analysis. Capture antibodies were covalently coupled to the surface of beads with unique fluorescence intensities (Becton Dickinson Biosciences). Detection antibodies were conjugated with R-Phycoerythrin (R-PE). A mixture of beads was firstly incubated with an appropriate standard mixture. Subsequently, a mixture of detection antibodies, either directly or indirectly conjugated to R-PE, was added to accomplish the desired sandwich format. The samples were finally analyzed on a BD FACSArrayTM Bioanalyzer. ELISAs were purchased from R&D Systems, PigCHAMP Pro Europe S.A. and ALPCO Diagnostics. Results and Conclusions : Table 1 shows validation parameters of the developed CBA 3-plex assay and the commercial ELISAs as mentioned by the manufacturer. Similar concentration-time profiles were observed for TNF-α, IL-1β, IL-6 and pig-MAP with CBA and ELISA in plasma from LPS-challenged pigs. Up till now, the CBA 2-plex assay could not be validated. On the other hand, the optimized and validated CBA 3-plex protocol provides a fast, flexible and cost-effective screening tool for simultaneous determination of pro-inflammatory cytokine profiles in porcine plasma. This technique will be applied in future research to study the immunomodulatory properties of drugs in a porcine LPS inflammation model.
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author
organization
year
type
conference
publication status
in press
subject
keyword
inflammation, pro-inflammatory cytokines, acute phase proteins, cytometric bead array (CBA), porcine plasma
in
JOURNAL OF VETERINARY PHARMACOLOGY AND THERAPEUTICS
J. Vet. Pharmacol. Ther.
conference name
12th International congress of the European Association for Veterinary Pharmacology and Toxicology (EAVPT)
conference location
Noordwijkerhout, The Netherlands
conference start
2012-07-08
conference end
2012-07-12
JCR category
VETERINARY SCIENCES
JCR impact factor
1.349 (2012)
JCR rank
43/142 (2012)
JCR quartile
2 (2012)
ISSN
0140-7783
language
English
UGent publication?
yes
classification
C3
additional info
uploaded document is presentation version
copyright statement
I have transferred the copyright for this publication to the publisher
id
2961705
handle
http://hdl.handle.net/1854/LU-2961705
date created
2012-07-12 14:07:34
date last changed
2012-08-09 14:50:34
@inproceedings{2961705,
  abstract     = {Introduction : Multiplex assays currently are a very popular tool for the simultaneous detection of biomarkers of infection and inflammation. Whereas specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single factor analysis, for multi-parameter analyses, this approach is time-consuming and expensive. Cytometric bead array (CBA) is a flexible, bead-based flow cytometric application for the simultaneous detection of various soluble proteins of interest. Therefore, the aim of the present study was to develop and validate a CBA 3-plex assay for the major pro-inflammatory cytokines TNF-\ensuremath{\alpha}, IL-1\ensuremath{\beta} and IL-6, and an additional CBA 2-plex assay for the major acute phase proteins (APPs) CRP and pig-MAP in plasma of lipopolysaccharide (LPS)-challenged pigs. Furthermore, results were compared to commercial ELISA kits. 
Materials and Methods : Four pigs with a mean body weight (BW) of 24.9 kg were intravenously challenged with 15 {\textmu}g ultrapure LPS/kg BW (Escherichia coli serotype O111:B4). Plasma was isolated and stored at -70{\textdegree}C until analysis. Capture antibodies were covalently coupled to the surface of beads with unique fluorescence intensities (Becton Dickinson Biosciences). Detection antibodies were conjugated with R-Phycoerythrin (R-PE). A mixture of beads was firstly incubated with an appropriate standard mixture. Subsequently, a mixture of detection antibodies, either directly or indirectly conjugated to R-PE, was added to accomplish the desired sandwich format. The samples were finally analyzed on a BD FACSArrayTM Bioanalyzer. ELISAs were purchased from R\&D Systems, PigCHAMP Pro Europe S.A. and ALPCO Diagnostics.
Results and Conclusions : Table 1 shows validation parameters of the developed CBA 3-plex assay and the commercial ELISAs as mentioned by the manufacturer. Similar concentration-time profiles were observed for TNF-\ensuremath{\alpha}, IL-1\ensuremath{\beta}, IL-6 and pig-MAP with CBA and ELISA in plasma from LPS-challenged pigs. Up till now, the CBA 2-plex assay could not be validated. On the other hand, the optimized and validated CBA 3-plex protocol provides a fast, flexible and cost-effective screening tool for simultaneous determination of pro-inflammatory cytokine profiles in porcine plasma. This technique will be applied in future research to study the immunomodulatory properties of drugs in a porcine LPS inflammation model.},
  author       = {Wyns, Heidi and Croubels, Siska and Demeyere, Kristel and Watteyn, Anneleen and De Backer, Patrick and Meyer, Evelyne},
  booktitle    = {JOURNAL OF VETERINARY PHARMACOLOGY AND THERAPEUTICS},
  issn         = {0140-7783},
  keyword      = {inflammation,pro-inflammatory cytokines,acute phase proteins,cytometric bead array (CBA),porcine plasma},
  language     = {eng},
  location     = {Noordwijkerhout, The Netherlands},
  title        = {Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines and acute phase proteins in porcine plasma},
  year         = {2012},
}

Chicago
Wyns, Heidi, Siska Croubels, Kristel Demeyere, Anneleen Watteyn, Patrick De Backer, and Evelyne Meyer. 2012. “Development of a Cytometric Bead Array Screening Tool for the Simultaneous Detection of Pro-inflammatory Cytokines and Acute Phase Proteins in Porcine Plasma.” In Journal of Veterinary Pharmacology and Therapeutics.
APA
Wyns, H., Croubels, S., Demeyere, K., Watteyn, A., De Backer, P., & Meyer, E. (2012). Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines and acute phase proteins in porcine plasma. JOURNAL OF VETERINARY PHARMACOLOGY AND THERAPEUTICS. Presented at the 12th International congress of the European Association for Veterinary Pharmacology and Toxicology (EAVPT).
Vancouver
1.
Wyns H, Croubels S, Demeyere K, Watteyn A, De Backer P, Meyer E. Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines and acute phase proteins in porcine plasma. JOURNAL OF VETERINARY PHARMACOLOGY AND THERAPEUTICS. 2012.
MLA
Wyns, Heidi, Siska Croubels, Kristel Demeyere, et al. “Development of a Cytometric Bead Array Screening Tool for the Simultaneous Detection of Pro-inflammatory Cytokines and Acute Phase Proteins in Porcine Plasma.” Journal of Veterinary Pharmacology and Therapeutics. 2012. Print.