Development of a sensitive and specific enzyme-linked immunosorbent assay for thymosin beta15, a urinary biomarker of human prostate cancer.

OBJECTIVES

In tissue-based assays, thymosin beta15 (Tbeta15) has been shown to correlate with prostate cancer (CaP) malignancy and with future recurrence. To be clinically effective, it must be shown that Tbeta15 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 Tbeta15 in human urine.

DESIGN AND METHODS

Sixteen antibodies were raised against recombinant Tbeta15 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 beta thymosins, was used to develop a competitive enzyme-linked immunosorbent assay (ELISA). Urinary Tbeta15 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 Tbeta15 signal was recovered from urine specimens stored at -20 degrees C for up to 1 year. At a threshold of 40 (ng/dL)/mug protein/mg creatinine), the assay had a sensitivity of 58% and a specificity of 94%. Relative to the control groups, Tbeta15 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 Tbeta15 at clinically relevant concentrations in urine from patients with CaP. The assay will provide a tool for future clinical trials to validate urinary Tbeta15 as a predictive marker for recurrent CaP.