title: Development of an ELISA to detect the secreted prostate cancer biomarker AGR2 in voided urine.
abstract: BACKGROUND
  Comparative transcriptomics between sorted cells identified AGR2 as one of the highest up-regulated genes in cancer. Overexpression in primary tumors was verified by tissue microarray analysis. AGR2 encodes a 19-kDa secreted protein that might be found in urine.
  
  
  METHODS
  Monoclonal antibodies were generated against AGR2. One antibody pair, P1G4 (IgG1) to capture and P3A5 (IgG2a) to detect, showed good performance characteristics in a sandwich ELISA. This assay could detect AGR2 at sub ng/ml quantities.
  
  
  RESULTS
  AGR2 was detected in tissue digestion media of tumor specimens and culture media of AGR2-secreting prostate cancer cell lines. Additional testings involved frozen section immunohistochemistry, immunoprecipitation, and Western blot analysis. Voided urine samples were collected from pre-operative cancer patients, and urinary protein was desalted and concentrated by filtration. The amount of AGR2 detected was scored as pg/100 µg total protein, and then converted to pg/ml urine. The developed ELISA detected AGR2 protein, ranging from 3.6 to 181 pg/ml, in an initial cohort of samples. AGR2 was not detected in the urine of non-cancer and a bladder cancer patient.
  
  
  CONCLUSIONS
  For prostate cancer, an AGR2 urine test could be used for diagnosis. The data, although derived from a small number of samples assayed, showed that developing such a test for clinical application is viable because AGR2 is specific to cancer cells, and apparently secreted into urine.
authors.affiliation: Antibody Resource, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Wayner
authors.forename: Sue-Ing
authors.initials: SI
authors.lastname: Quek
authors.forename: Rumana
authors.initials: R
authors.lastname: Ahmad
authors.forename: Melissa E
authors.initials: ME
authors.lastname: Ho
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Loprieno
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhou
authors.forename: William J
authors.initials: WJ
authors.lastname: Ellis
authors.forename: Lawrence D
authors.initials: LD
authors.lastname: True
authors.forename: Alvin Y
authors.initials: AY
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000378:metabolism* / Q000652:urine*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009088:Mucoproteins; D015513:Oncogene Proteins; D011471:Prostatic Neoplasms / Q000737:chemistry / Q000378:metabolism* / Q000652:urine*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: C502296:AGR2 protein, human; D014408:Biomarkers, Tumor; D009088:Mucoproteins; D015513:Oncogene Proteins; D011506:Proteins