Microplate magnetic chemiluminescence immunoassay for detecting urinary survivin in bladder cancer

Abstract

Survivin is a tumor marker for bladder cancer; however the role of urinary survivin levels has not been fully elucidated due to the limitations of current detection methods. Based on two survivin-specific monoclonal antibodies (McAbs) already confirmed through enzyme linked immunosorbent assays, the present study aimed to establish a microplate magnetic chemiluminescence immunoassay (CLIA) for the detection of urinary survivin levels and evaluate its application for the diagnosis of patients with bladder cancer. Horseradish peroxidase and biotin conjugates were used to label two different anti-survivin McAbs, respectively. The labeled antibodies combined with survivin to form a sandwiched immune complex. The streptavidin magnetic particles (MPs) served as the solid phase and the separator. The relevant parameters involved in the immunoassay, including the immunoassay reagents used and the physicochemical parameters were optimized. Then, urine samples from 130 patients with bladder cancer and 113 healthy controls were detected, and analyzed using the established method. The method was linear to 1,000 ng/ml survivin with a detection limit of 0.83 ng/ml. The intra- and inter-assay coefficients of variation were <8, and <11%, respectively. The concentration of diluted survivin and the dilution ratios gave a linear correlation of 0.9989. The results demonstrated that the urinary survivin levels in patients with bladder cancer were significantly higher (P<0.001) compared with that in healthy controls. At a survivin concentration of 2.0884 ng/ml, the sensitivity and specificity were 86.9 and 61.9%, respectively. Furthermore, the urinary survivin levels were positively correlated with metastatic stage, histological stage and recurrence (P<0.01). In conclusion, the present study preliminarily proposed a microplate magnetic CLIA for survivin detection and further evaluated the value of urinary survivin as a diagnostic marker for bladder cancer.

Keywords: bladder cancer; microplate magnetic chemiluminescence immuno-assay; survivin; tumor markers.

Figure 1.

Schematic illustration of the established MPs-CLIA method. MPs, magnetic particles.

Figure 2.

Determination of the best antibody pair. Detection methods: S/B ratio obtained for both antibody pairs, with 10, 100 and 1,000 ng/ml of the survivin standards. RLU, relative light unit.

Figure 3.

Influence of the dilution ratios of the HRP-labeled antibody and the biotinylated antibody. Detection conditions: the standard survivin concentration of 100 ng/ml; room temperature; 1 µl of streptavidin-coated MPs and 150 µl of CL substrate. The four curves correspond to a series of dilution ratios of the HRP-labeled antibody. *P<0.05. RLU, relative light unit; MPs, magnetic particles.

Figure 4.

Optimization of the volume of MPs. Detection conditions: The standard survivin concentration is 50 ng/ml; room temperature and 150 µl of CL substrate; the HRP-labeled antibody dilution ratio is 1:15,000; the biotinylated antibody dilution ratio is 1:18,000. *P=0.01. RLU, relative light unit; MPs, magnetic particles.

Figure 5.

Influence of substrate volume and time required for enzyme-substrate incubation on RLU values. Detection conditions: a standard positive sample (50 ng/ml) was used to evaluate the effect; room temperature and 1 µl of Streptavidin-coated MPs; the HRP-labeled antibody dilution ratio is 1:15,000; the biotinylated antibody dilution ratio is 1:18,000. The six plots correspond to a series of durations required for enzyme-substrate incubation (10,20,30,40,50 and 60 min). RLU: *P<0.05. RLU, relative light unit; MPs, magnetic particles.

Figure 6.

Calibration curve for survivin standards. The calibration curve was constructed under the optimized conditions and showed a detection range from 0.97 to 1,000 ng/ml with a detection limit of 0.83 ng/ml. RLU, relative light unit.

Figure 7.

The linearity-dilution effect of the high concentration sample.

Figure 8.

Correlation between urinary survivin levels measured by the newly established method and ELISA.

Figure 9.

ROC curve. Survivin levels of urine samples were detected among healthy individuals and bladder cancer patients. ROC, receiver operating characteristic.

Figure 10.

Survivin levels in healthy controls and bladder cancer patients. The urine of 113 healthy controls and 130 bladder cancer patients were detected.