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

BMC Biotechnology

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

[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

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

Others  :  1085006
DOI  :  10.1186/1472-6750-14-63

 received in 2014-04-06, accepted in 2014-07-10,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Altara et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Motilva V: Inflammation and cancer: new targets and novel therapeutic approach. Curr Pharm Des 2012, 18(26):3829-3830.
• [2]Corsten MF, Schroen B, Heymans S: Inflammation in viral myocarditis: friend or foe? Trends Mol Med 2012, 18(7):426-437.
• [3]Lam HS, Ng PC: Biochemical markers of neonatal sepsis. Pathology 2008, 40(2):141-148.
• [4]Ahmad T, Fiuzat M, Felker GM, O’Connor C: Novel biomarkers in chronic heart failure. Nat Rev Cardiol 2012, 9(6):347-359.
• [5]Leng SX, McElhaney JE, Walston JD, Xie D, Fedarko NS, Kuchel GA: ELISA and multiplex technologies for cytokine measurement in inflammation and aging research. J Gerontol A Biol Sci Med Sci 2008, 63(8):879-884.
• [6]Lequin RM: Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem 2005, 51(12):2415-2418.
• [7]Liu MY, Xydakis AM, Hoogeveen RC, Jones PH, Smith EO, Nelson KW, Ballantyne CM: Multiplexed analysis of biomarkers related to obesity and the metabolic syndrome in human plasma, using the Luminex-100 system. Clin Chem 2005, 51(7):1102-1109.
• [8]Haab BB, Zhou H: Multiplexed protein analysis using spotted antibody microarrays. Methods Mol Biol 2004, 264:33-45.
• [9]van Eickels M, Grohe C, Cleutjens JP, Janssen BJ, Wellens HJ, Doevendans PA: 17beta-estradiol attenuates the development of pressure-overload hypertrophy. Circulation 2001, 104(12):1419-1423.
• [10]Wass JA: R: a language and environment for statistical computing. R Found Stat 2012, Version 2.11.1 (2010-05-31):1.
• [11]Christia P, Bujak M, Gonzalez-Quesada C, Chen W, Dobaczewski M, Reddy A, Frangogiannis NG: Systematic characterization of myocardial inflammation, repair, and remodeling in a mouse model of reperfused myocardial infarction. J Histochem Cytochem 2013, 61(8):555-570.
• [12]Coggins M, Rosenzweig A: The fire within: cardiac inflammatory signaling in health and disease. Circ Res 2012, 110(1):116-125.
• [13]Braunwald E: Biomarkers in heart failure. N Engl J Med 2008, 358(20):2148-2159.