The long-term objectives of the proposed study are to develop a sensitive immunoassay which can detect the presence and onset of benign prostate hyperplasia (BPH), quantify progression of BPH growth, and differentiate BPH from prostate cancer (CaP). A biomarker that can distinguish benign from malignant prostate disease and a biomarker that can predict and monitor progression of MPH would have a major impact on reducing the problem of overtreating and high cost of healthcare for BPH in the United States.
The specific aims of this application are (l) To identify a BPH biomarker in seminal plasma or prostate fluid, and (2) To develop an immunoassay to detect and quantify the BPH biomarker in serum, urine and/or seminal plasma. Seminal plasma and prostate fluid will be used to begin the search for a specific or highly. restricted BPH biomarker because these body fluids should contain the highest concentration of secreted molecules of prostate origin (especially prostate fluid) without being significantly contaminated with molecules originating from other parts of the body. Three different approaches will be used to enrich the chances of identifying rare proteins that are unique or novel to BPH. All 3 approaches will lead to the production of specific monoclonal antibodies (MoAb). For the first approach, seminal plasma or prostate fluid will be immunodepleted of irrelevant proteins and then separated by two- dimensional electrophoresis. Computerized protein profiles for BPH will be compared with profiles of seminal plasma from normal donors and patients with either prostatitis or CaP. Any unique proteins identified in the BPH profile will be eluted and used to produce MoAbs. Tolerance induction is the second approach. Neonatal mice will be rendered tolerant to normal prostate and CaP antigens, and then immunized with the immunodepleted and enriched BPH immunogens in an effort to select BPH specific immune clones for production of MoAbs. Subtractive hybridization, the third approach, assumes that mRNA transcripts exist that are characteristic of the BPH phenotype. To detect mRNAs differentially expressed in BPH and not in normal or malignant prostate, a subtracted cDNA library will be differentially screened with BPH, CaP and normal cDNA probes. BPH cDNAs identified by Northern blot analysis are expressed as recombinant proteins. Polyclonal antibodies against the recombinant proteins will then be used to screen prostate fluids to identify unique BPH proteins. Recombinant proteins found to be unique for BPH will be used to produce MoAbs. The most specific MoAbs obtained by these 3 approaches will be used to develop a sensitive 2-site immunoradiometric assay to quantify BPH biomarkers in serum, urine and prostate fluid.
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