Improving membrane based multiplex immunoassays for semi-quantitative detection of multiple cytokines in a single sample

Raffaele Altara*, Marco Manca, Marleen H. M. Hessel, Ben J. Janssen, Harry H. A. Struijker-Boudier, Rob J. J. Hermans, Matthijs Blankesteijn

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

9 Citations (Web of Science)
10 Downloads (Pure)

Abstract

Background: Inflammatory mediators can serve as biomarkers for the monitoring of the disease progression or prognosis in many conditions. In the present study we introduce an adaptation of a membrane-based technique in which the level of up to 40 cytokines and chemokines can be determined in both human and rodent blood in a semi-quantitative way. The planar assay was modified using the LI-COR (R) detection system (fluorescence based) rather than chemiluminescence and semi-quantitative outcomes were achieved by normalizing the outcomes using the automated exposure settings of the Odyssey readout device. The results were compared to the gold standard assay, namely ELISA. Results: The improved planar assay allowed the detection of a considerably higher number of analytes (n = 30 and n = 5 for fluorescent and chemiluminescent detection, respectively). The improved planar method showed high sensitivity up to 17 pg/ml and a linear correlation of the normalized fluorescence intensity with the results from the ELISA (r = 0.91). Conclusions: The results show that the membrane-based technique is a semi-quantitative assay that correlates satisfactorily to the gold standard when enhanced by the use of fluorescence and subsequent semi-quantitative analysis. This promising technique can be used to investigate inflammatory profiles in multiple conditions, particularly in studies with constraints in sample sizes and/or budget.
Original languageEnglish
Article number63
JournalBMC Biotechnology
Volume14
DOIs
Publication statusPublished - 15 Jul 2014

Keywords

  • Cytokines
  • Chemokines
  • Multiplex assay
  • Planar assay
  • Bead-based assay
  • Inflammatory mediators

Cite this