title: MicroRNA in vitro diagnostics using immunoassay analyzers.
abstract: BACKGROUND
  The implementation of new biomarkers into clinical practice is one of the most important areas in medical research. Besides their clinical impact, novel in vitro diagnostic markers promise to have a substantial effect on healthcare costs. Although numerous publications report the discovery of biomarkers, only a fraction of those markers are routinely used. One key challenge is a measurement system that is compatible with clinical workflows.
  
  
  METHODS
  We designed a new immunoassay for microRNA (miRNA) quantification. The assay combines streptavidin-linked microparticles, a biotinylated catcher oligonucleotide complementary to a single miRNA species, and finally, a monoclonal antibody to DNA/RNA heterohybrids labeled with acridinium ester. Importantly, our assay runs on standard immunoassay analyzers. After a technical validation of the assay, we evaluated the clinical performance on 4 Alzheimer disease miRNAs.
  
  
  RESULTS
  Our assay has an analytical specificity of 99.4% and is at the same time sensitive (concentrations in the range of 1 pmol/L miRNA can be reliably profiled). Because the novel approach did not require amplification steps, we obtained high reproducibility for up to 40 biological replicates. Importantly, our assay prototype exhibited a time to result of <3 h. With human blood samples, the assay was able to measure 4 miRNAs that can detect Alzheimer disease with a diagnostic accuracy of 82% and showed a Pearson correlation >0.994 with the gold standard qRT-PCR.
  
  
  CONCLUSIONS
  Our miRNA immunoassay allowed the measurement of miRNA signatures with sufficient analytical sensitivity and high specificity on commonly available laboratory equipment.
authors.affiliation: Corporate Technology, Siemens AG, Erlangen, Germany;
authors.forename: Andreas
authors.initials: A
authors.lastname: Kappel
authors.affiliation: Clinical Bioinformatics, Medical Faculty, Saarland University, and.
authors.forename: Christina
authors.initials: C
authors.lastname: Backes
authors.affiliation: Corporate Technology, Siemens AG, Erlangen, Germany;
authors.forename: Yiwei
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Clinical Bioinformatics, Medical Faculty, Saarland University, and.
authors.forename: Sachli
authors.initials: S
authors.lastname: Zafari
authors.affiliation: Department of Human Genetics, Saarland University, Saarbrücken, Germany;
authors.forename: Petra
authors.initials: P
authors.lastname: Leidinger
authors.affiliation: Internal Medicine III, University Hospital Heidelberg, Heidelberg, Germany;
authors.forename: Benjamin
authors.initials: B
authors.lastname: Meder
authors.affiliation: Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany.
authors.forename: Herbert
authors.initials: H
authors.lastname: Schwarz
authors.affiliation: Corporate Technology, Siemens AG, Erlangen, Germany;
authors.forename: Walter
authors.initials: W
authors.lastname: Gumbrecht
authors.affiliation: Department of Human Genetics, Saarland University, Saarbrücken, Germany;
authors.forename: Eckart
authors.initials: E
authors.lastname: Meese
authors.affiliation: Strategy, Siemens Healthcare, Erlangen, Germany;
authors.forename: Cord F
authors.initials: CF
authors.lastname: Staehler
authors.affiliation: Clinical Bioinformatics, Medical Faculty, Saarland University, and andreas.keller@ccb.uni-saarland.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Keller
mesh_terms: D000544:Alzheimer Disease / Q000097:blood / Q000235:genetics*; D000911:Antibodies, Monoclonal; D015415:Biomarkers / Q000097:blood*; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D035683:MicroRNAs / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D035683:MicroRNAs