Prostate cancer has become a formidable problem for the medical communities of developed countries including the U.S.A. As the U.s. population ages, the prevalence of the disease is increasing even more rapidly. Although PSA testing has aided in diagnosis of the disease greatly, more sensitive and accurate assays are required urgently. The purposes of this proposal are to develop and evaluate new immunoassay and molecular detection systems for early detection and prognosis of prostate cancer. Monospecific antibodies to human glandular kallikrein (hK2) will be used to develop a new immunoassay. hK2 is similar to PSA in its physiochemical properties and has 75% identify to PSA. The close homology and expression mode of these two proteins suggest that both proteins may provide valuable information, either independently or as a ratio, in the detection of prostate cancer. We propose detailed experiments for production of recombinant hK2 protein, purification of hK2 proteins (recombinant and endogenous), and full characterization of biochemical and immunologic properties of the proteins. Polyclonal and monoclonal antibodies specific for different epitopes of hK2 will be generated and characterized by using synthetic hK2 peptides, recombinant hK2 and purified hK2 from natural sources. pure hK2 protein will be obtained from heterologous expression systems (bacteria, yeast, baculovirus, etc.) and prostate tissue and seminal plasma as assay reagents and immunogenes. Highly sensitive assay system for hK2 using chemiluminescent signal in conjunction with competitive or sandwhich assays will be developed. Once an effective immunoassay is developed, a pilot study will be performed using serum specimens including those previously used in the studies of PSA with a stratified sample of 471 men from Olmsted County population without prostate cancer and a collection of 300 specimens from men with various stages of untreated prostate cancer. In addition, we plan to collect specimens from approximately 200 native Alaskans to determine the distribution of both hK2 and PSA. The hK2 values also will be analyzed to determine the correlation and association with total, free and complex PSA. Recently, it has been shown that the reverse-transcriptase polymerase chain reaction (RT-PCR) for PSA mRNA can be used to detect micrometastasis of prostate cancer. Because of the heterogeneity nature of advanced prostate cancer, this proposal will use multiple human prostate-specific genes as RT-PCR targets in order to develop a valid system for early detection of prostate cancer cells with metastasis potential in distant body compartments. The accomplishment of this entire proposal will not only produce new sensitive and specific detection systems for prostate cancer but also provide tremendous new information regarding natural history and tumor biology of the disease.
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