Development of an ELISA to detect the secreted prostate cancer biomarker AGR2 in voided urine.

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.