title: Development of a sensitive and specific enzyme-linked immunosorbent assay for thymosin beta15, a urinary biomarker of human prostate cancer.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Program in Vascular Biology and Department of Surgery, Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Lloyd M
authors.initials: LM
authors.lastname: Hutchinson
authors.forename: Eric L
authors.initials: EL
authors.lastname: Chang
authors.forename: Christian M
authors.initials: CM
authors.lastname: Becker
authors.forename: Naoko
authors.initials: N
authors.lastname: Ushiyama
authors.forename: Dani
authors.initials: D
authors.lastname: Behonick
authors.forename: Mei-Chiung
authors.initials: MC
authors.lastname: Shih
authors.forename: William C
authors.initials: WC
authors.lastname: DeWolf
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Gaston
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Zetter
mesh_terms: D000595:Amino Acid Sequence; D014408:Biomarkers, Tumor / Q000652:urine*; D016022:Case-Control Studies; D016384:Consensus Sequence; D017124:Conserved Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D011471:Prostatic Neoplasms / Q000517:prevention & control* / Q000532:radiotherapy / Q000652:urine*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000652:urine; D012008:Recurrence; D012680:Sensitivity and Specificity; D013947:Thymosin / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000652:urine*
chemical_list: D014408:Biomarkers, Tumor; D011994:Recombinant Proteins; D013947:Thymosin