One More Bottleneck towards Biomarker Validation and Clinical Implementation

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

Analytical Performance of ELISA Assays in Urine: One More Bottleneck towards Biomarker Validation and Clinical Implementation.

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

Study of the Total Phenolic Content, Total Antioxidant Activity and In Vitro Digestibility of Novel Wheat Crackers Enriched with Cereal, Legume and Agricultural By-Product Flours

Wheat-flour crackers represent a staple snack option, although they lack nutritional value. Agricultural by-products such as olive and grape seeds, cereals such as barley and legumes such as lupine and chickpea are rich in bioactive compounds; therefore, flours obtained from those could represent a better option for bakery products fortification. The purpose of the present study was the investigation of total phenolic content and antioxidant activity before and after the baking of wheat crackers enriched with 10–30% olive seed, 10–30% grape seed, 10–40% lupine, 10–30% barley and 20–60% and 80% chickpea flours and the evaluation of the predicted bioavailability after in vitro digestion of crackers demonstrating the highest values. Crackers and doughs were processed and analyzed using Folin–Ciocâlteu and ferric reducing antioxidant power (FRAP) assays, respectively. Crackers with the highest properties were subjected to in vitro gastrointestinal digestion. Baking resulted in an increase in total phenolics and antioxidant activity in the majority of crackers. Olive and grape seed flour crackers demonstrated the highest antioxidant properties. Following in vitro digestion, 30% olive seed flour crackers retained the majority of polyphenols and antioxidant activity. Crackers enriched with 30% olive seed flour could represent a healthy functional bakery snack regarding their increased antioxidant properties

Summary of analytical performance.

<p>Summary of analytical performance.</p

Linearity results of A) SPARC and B) PR3.

<p>For each biomarker a high concentration sample was serially diluted and theoretical values were compared to the experimental.</p

Recovery study results of PR3. Negative urine samples were spiked with low, medium and high concentration of standard.

<p>Recovery study results of PR3. Negative urine samples were spiked with low, medium and high concentration of standard.</p

Reproducibility study results of A) SPARC and B) PR3.

<p>Three urine samples with low, medium and high concentration were used.</p

Standard curve validation of A) SPARC (R² = 0.999) and B) PR3 (R² = 0.996).

<p>Standard curve validation of A) SPARC (R² = 0.999) and B) PR3 (R² = 0.996).</p

Recovery study results of SPARC. Negative urine samples were spiked with low, medium and high concentration of standard.

<p>Recovery study results of SPARC. Negative urine samples were spiked with low, medium and high concentration of standard.</p