Development of a sensitive and specific enzyme-linked immunosorbent assay for thymosin β15, a urinary biomarker of human prostate cancer

Objectives: In tissue-based assays, thymosin β15 (Tβ15) has been shown to correlate with prostate cancer (CaP) malignancy and with future recurrence. To be clinically effective, it must be shown that Tβ15 is released by the tumor into body fluids in detectable concentrations. Toward this end, we have worked to develop a quantitative high-throughput assay that can accurately measure clinically relevant concentrations of Tβ15 in human urine. Design and methods: Sixteen antibodies were raised against recombinant Tβ15 and/or peptide conjugates. One antibody, having stable characteristics over the wide range of pH and salt concentrations found in urine and minimal cross-reactivity with other β thymosins, was used to develop a competitive enzyme-linked immunosorbent assay (ELISA). Urinary Tβ15 concentration was determined for control groups; normal (N = 52), prostate intraepithelial neoplasia (PIN, N = 36), and CaP patients; untreated (N = 7) with subsequent biochemical failure, radiation therapy (N = 17) at risk of biochemical recurrence. Results: The operating range of the competition ELISA fell between 2.5 and 625 ng/mL. Recoveries exceeded 75%, and the intra- and inter-assay coefficients of variability were 3.3% and 12.9%, respectively. No cross-reactivity with other urine proteins was observed. A stable Tβ15 signal was recovered from urine specimens stored at −20°C for up to 1 year. At a threshold of 40 (ng/dL)/μg protein/mg creatinine), the assay had a sensitivity of 58% and a specificity of 94%. Relative to the control groups, Tβ15 levels were greater than this threshold in a significant fraction of the CaP patients (P< 0.001), including 5 of the 7 patients who later experienced PSA recurrence. Conclusions: We have established an ELISA that is able to detect Tβ15 at clinically relevant concentrations in urine from patients with CaP. The assay will provide a tool for future clinical trials to validate urinary Tβ15 as a predictive marker for recurrent CaP.