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Open Access Methodology article

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

Raffaele Altara1*, Marco Manca2, Marleen HM Hessel1, Ben J Janssen1, Harry H A Struijker-Boudier1, Rob JJ Hermans1 and W Matthijs Blankesteijn1

Author Affiliations

1 Department of Pharmacology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands

2 Department of Pathology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands

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BMC Biotechnology 2014, 14:63  doi:10.1186/1472-6750-14-63

Published: 15 July 2014

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.

Keywords:
Cytokines; Chemokines; Multiplex assay; Planar assay; Bead-based assay; Inflammatory mediators