title: Improving membrane based multiplex immunoassays for semi-quantitative detection of multiple cytokines in a single sample.
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.
authors.affiliation: Department of Pharmacology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands. r.altara@maastrichtuniversity.nl.
authors.forename: Raffaele
authors.initials: R
authors.lastname: Altara
authors.forename: Marco
authors.initials: M
authors.lastname: Manca
authors.forename: Marleen H M
authors.initials: MH
authors.lastname: Hessel
authors.forename: Ben J
authors.initials: BJ
authors.lastname: Janssen
authors.forename: Harry H A
authors.initials: HH
authors.lastname: Struijker-Boudier
authors.forename: Rob J J
authors.initials: RJ
authors.lastname: Hermans
authors.forename: W Matthijs
authors.initials: WM
authors.lastname: Blankesteijn
mesh_terms: D000818:Animals; D016207:Cytokines / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D053583:Interleukin-1beta / Q000032:analysis; D008163:Luminescent Measurements; D008297:Male; D008567:Membranes, Artificial; D051379:Mice; D020543:Proteome / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D014409:Tumor Necrosis Factor-alpha / Q000097:blood
chemical_list: D016207:Cytokines; D053583:Interleukin-1beta; D008567:Membranes, Artificial; D020543:Proteome; D014409:Tumor Necrosis Factor-alpha