title: Analytical Performance of ELISA Assays in Urine: One More Bottleneck towards Biomarker Validation and Clinical Implementation.
abstract: ELISA is the main approach for the sensitive quantification of protein biomarkers in body fluids and is currently employed in clinical laboratories for the measurement of clinical markers. As such, it also constitutes the main methodological approach for biomarker validation and further qualification. For the latter, specific assay performance requirements have to be met, as described in respective guidelines of regulatory agencies. Even though many clinical ELISA assays in serum are regularly used, ELISA clinical applications in urine are significantly less. The scope of our study was to evaluate ELISA assay analytical performance in urine for a series of potential biomarkers for bladder cancer, as a first step towards their large scale clinical validation. Seven biomarkers (Secreted protein acidic and rich in cysteine, Survivin, Slit homolog 2 protein, NRC-Interacting Factor 1, Histone 2B, Proteinase-3 and Profilin-1) previously described in the literature as having differential expression in bladder cancer were included in the study. A total of 11 commercially available ELISA tests for these markers were tested by standard curve analysis, assay reproducibility, linearity and spiking experiments. The results show disappointing performance with coefficients of variation>20% for the vast majority of the tests performed. Only 3 assays (for Secreted protein acidic and rich in cysteine, Survivin and Slit homolog 2 protein) passed the accuracy thresholds and were found suitable for further application in marker quantification. These results collectively reflect the difficulties in developing urine-based ELISA assays of sufficient analytical performance for clinical application, presumably attributed to the urine matrix itself and/or presence of markers in various isoforms.
authors.affiliation: Biotechnology Laboratory, Centre of Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
authors.forename: Despina
authors.initials: D
authors.lastname: Chatziharalambous
authors.affiliation: Biotechnology Laboratory, Centre of Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
authors.forename: Vasiliki
authors.initials: V
authors.lastname: Lygirou
authors.affiliation: Biotechnology Laboratory, Centre of Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.|Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Latosinska
authors.affiliation: Department of Urology, Laikon Hospital, University of Athens, School of Medicine, Athens, Greece.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Stravodimos
authors.affiliation: Biotechnology Laboratory, Centre of Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
authors.forename: Antonia
authors.initials: A
authors.lastname: Vlahou
authors.affiliation: RWTH-Aachen, Institute for Molecular Cardiovascular Research (IMCAR), Aachen, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Jankowski
authors.affiliation: Biotechnology Laboratory, Centre of Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
authors.forename: Jerome
authors.initials: J
authors.lastname: Zoidakis
mesh_terms: D014408:Biomarkers, Tumor / Q000652:urine*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015203:Reproducibility of Results; D001749:Urinary Bladder Neoplasms / Q000652:urine*
chemical_list: D014408:Biomarkers, Tumor