Exploring solid-phase proximity ligation assay for survivin detection in urine

Abstract

Urine-based biomarkers are a rational and promising approach for the detection of bladder cancer due to the proximity of urine to the location of the tumor site and the non-invasive nature of its sampling. A well-known and highly investigated biomarker for bladder cancer is survivin. For detection of very small amounts of urinary survivin protein a highly sensitive assay was developed. The assay is based on the immuno-PCR technology, more precisely a solid-phase proximity ligation assay (spPLA). The limit of detection for the survivin spPLA was 1.45 pg/mL, resulting in an improvement of the limit of detection by a factor of approximately 23 compared to the previously in-house developed survivin ELISA. A key step in development was the initial isolation of survivin by a molecular fishing rod based on magnetic beads. Interfering matrix compounds pose a special challenge for further analytical application, but can be overcome by this isolation step. The assay is designed to work with only 500 μL of voided urine. The survivin spPLA showed a sensitivity of 30% and specificity of 89% for bladder cancer detection in this study of 110 bladder cancer cases and 133 clinical controls. Moreover, the results demonstrated again that survivin is a useful complementary marker in combination with UBC® Rapid by increasing the overall sensitivity to 70% with a specificity of 86%. Although the performance for detection of bladder cancer was rather low, the herein developed assay might serve as a new tool for survivin biomarker research in diverse human fluids, even if the biological matrix is complex or survivin is only present in small amounts.

Fig 1. Reference curve of the survivin spPLA based on 40 independent experiments.

Survivin concentrations ranged from 0.32 pg/mL to 5000 pg/mL. (a) Raw Ct-values versus survivin concentrations. (b) Delta Ct-values versus survivin concentrations. (c) Comparison of spPLA (solid) and ELISA (dotted, mean of 44 independent experiments) for survivin detection.

Fig 2. Workflow for spPLA detection of survivin in urinary samples.

After urine concentration the remaining sample is diluted in buffer for pH neutralization. After sequential binding of survivin antibody-functionalized magnetic beads and proximity probes, DNA-strands come into close proximity. A connector allows the ligation, thereby forming a new chimeric DNA strand. Real-time PCR and further analysis allow protein quantification.

Fig 3. Group analysis of spPLA survivin measurements.

Detectable amounts of survivin are depicted as dot plots on a logarithmic scale for cases and clinical controls with median and range. Samples containing no measurable amounts of survivin are indicated with 0*. LoD: Limit of detection.

Fig 4. Venn diagram for survivin spPLA and UBC Rapid for both groups, cases and clinical controls.

The cutoff for the spPLA was 1.45 pg/mL, resulting in 33 positive cases and 14 positive clinical controls. The cutoff for UBC Rapid assay was set to 10 mg/L, resulting in 61 positive cases and 5 positive clinical controls.