title: Protein microarray platform for the multiplex analysis of biomarkers in human sera.
abstract: Many biomarkers are currently used to monitor patients in clinical studies. Technologies which evaluate, validate and monitor biomarkers in a cost effective and efficient manner are a necessity. Here we describe the development, validation and implementation of a protein microarray platform for the quantitative and simultaneous analysis of six proteins: IL-1beta, IL-1ra, IL-6, IL-8, MCP-1 and TNFalpha. The platform utilizes a 96-well plate as a solid support on which antibodies are immobilized using non-contact piezoelectric printing. The reaction is based on a sandwich ELISA and the signal is quantified by chemiluminescence with a CCD camera. The robustness and reproducibility of the methodology was investigated using the Food and Drug Administration (FDA) regulatory guidelines for pharmacokinetic assay validation, in which a spike-recovery validation test was elaborated and run over 3 days. The method was shown to be both quantitative and reproducible, with assay accuracy between 70% and 130%, and an assay precision of less than 30%. In addition, protein microarray performance was compared with the classical ELISA approach. Sera collected from a total of 78 individuals were assayed using both approaches. Correlation coefficients (R2) between the two technologies were calculated for each of the analytes: 0.90 for IL-1beta, 0.60 for IL-1ra, 0.93 for IL-6, 0.96 for IL-8, 0.94 for MCP-1 and 0.95 for TNFalpha. The results obtained demonstrate the applicability of this protein microarray for quantitative and simultaneous analysis of IL-1beta, IL-1ra, IL-6, IL-8, MCP-1 and TNFalpha in clinical samples.
authors.affiliation: Novartis Pharma AG Werk Klybeck, Postfach, CH-4002, Basel, Switzerland.
authors.forename: Teresa
authors.initials: T
authors.lastname: Urbanowska
authors.forename: Sara
authors.initials: S
authors.lastname: Mangialaio
authors.forename: Christophe
authors.initials: C
authors.lastname: Zickler
authors.forename: Sirinda
authors.initials: S
authors.lastname: Cheevapruk
authors.forename: Paul
authors.initials: P
authors.lastname: Hasler
authors.forename: Stephan
authors.initials: S
authors.lastname: Regenass
authors.forename: Francois
authors.initials: F
authors.lastname: Legay
mesh_terms: D015415:Biomarkers / Q000097:blood*; D018932:Chemokine CCL2 / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein / Q000097:blood; D053583:Interleukin-1beta / Q000097:blood; D015850:Interleukin-6 / Q000097:blood; D016209:Interleukin-8 / Q000097:blood; D040081:Protein Array Analysis / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000097:blood
chemical_list: D015415:Biomarkers; C111835:CCL2 protein, human; D018932:Chemokine CCL2; D053590:Interleukin 1 Receptor Antagonist Protein; D053583:Interleukin-1beta; D015850:Interleukin-6; D016209:Interleukin-8; D014409:Tumor Necrosis Factor-alpha