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
