Purified human prostate antigen

Abstract

A prostate antigen distinct from prostatic acid phosphatase has been detected in normal, benign hypertrophic and malignant prostatic tissues, but not in other human tissues. The prostate antigen was purified to homogeneity from prostatic tissues by ammonium sulfate precipitation, DEAE-BioGel A anion exchange chromatography, molecular sievings on Sephadex G-100 and Sephadex G-75, This invention was supported in part by Grants No. CA-15126 and CA-15437 from the National Cancer Institute, U.S. Public Health Service.

What is claimed is: 
     
       1. An in vivo composition of matter comprising an immunochemically effective concentration and amount of a purified human prostate specific antigen occurring in normal and cancerous prostatic tissue and purified to show a single protein band on analytical polyacrylamide gel electrophoresis and isoelectric focusing, said antigen having a molecular weight as determined by Sephadex G-75 gel of filtration of about 33,000 and by a molecular weight as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis of about 34,000 with no subunit and having an isoelectric point pI of 6.9, said antigen further being insoluble in perchloric acid and substantially free of normal serum protein components, water-insoluble cellular material and of prostatic acid phosphatases. 
     
     
       2. An in vitro composition of matter comprising an immunochemically effective concentration and amount of antibodies against a human prostate specific antigen occurring in normal and cancerous prostatic tissue and purified to show a single protein band on analytical polyacrylamide gel electrophoresis and isoelectric focusing, said antigen having a molecular weight by Sephadex G-75 gel filtration of about 33,000 and by sodium dodecyl sulfate polyacrylamide gel electrophoresis of about 34,000 with no subunit and having an isoelectric point pI of 6.9, said antigen further being insoluble in perchloric acid and said composition being free of antibodies against humen prostatic acid phosphatases. 
     
     
       3. A composition according to claim 2, wherein said antibodies are monoclonal antibodies. 
     
     
       4. A composition according to claim 2, wherein said antibodies are immunoprecipitating antibodies. 
     
     
       5. A composition according to claim 2, wherein said antibodies are labeled for radioimmunoassay or enzyme-linked immunoassay. 
     
     
       6. A composition according to claim 2, wherein said antibodies are covalently bonded to a water-insoluble support. 
     
     
       7. A process for preparing immunoprecipitating antibodies to antigens associated with prostatic tissue and fluid, which comprises: (a) extracting an antigen according to claim 1 from prostatic tissue or separating said antigen from prostatic fluid;   (b) separating said antigen from extraneous antigenic proteins to obtain a composition consisting essentially of said antigen;   (c) immunizing animals with the resultant purified antigen to form antibodies specific thereto; and   (d) recovering immunoprecipitating antibodies against said antigen from said animals.   
     
     
       8. A process according to claim 7, wherein said antigen is separated from extraneous antigenic protein material by salting out. 
     
     
       9. A process according to claim 7, wherein said antigen is obtained from seminal fluid. 
     
     
       10. A process preparing antibodies to antigens associated with prostatic tissue and fluid, which comprises: (a) cloning hybridoma cells capable of secreting said antibodies;   (b) extracting antibodies according to claim 2 from said secretions;   (c) separating said antibodies from extraneous antigenic proteins to obtain a composition consisting essentially of said antibodies; and   (d) recovering an immunologically effective concentration and amount of said antibodies from said cells.   
     
     
       11. A process according to claim 10, wherein said hybridoma cells are prepared by fusing a nonsecretory myeloma cell line with primary mouse spleen cells. 
     
     
       12. A method for diagnosing carcinoma of the prostate, which comprises: (a) forming an immunoprecipitin complex between (i) an an antigen consisting essentially of the human prostate specific antigen of claim 1 associated with both normal and cancerous prostatic tissue which is distinct from prostatic acid phosphatase and (ii) immunoprecipitating antibodies thereto which are free of cross-reactivity against prostatic acid phosphatases and against antigens associated with other carcinomas; and   (b) detecting the presence of said complex.   
     
     
       13. A method according to claim 12, wherein the complex is formed by countercurrent immunoelectrophoresis. 
     
     
       14. A method according to claim 12, wherein the complex is formed by immunologically reacting said antigen with antibodies thereto covalently bound to a water-insoluble carrier. 
     
     
       15. A method according to claim 12, wherein said complex is detected by radioimmunoassay or by enzyme-linked immunoassay. 
     
     
       16. A continuous murine cell line capable of producing monoclonal antibodies of the IgM isotype to the specific antigen of claim 1 under nutrient growth conditions, consisting essentially of a fused cell hybrid of: (a) primed antibody-producing cells with   (b) myeloma cells capable of producing a homogeneous population of immunoglobulin in the fused hybridoma.   
     
     
       17. A cell line according to claim 16, wherein the fused cell hybrid is ATCC HB 8051. .Iadd.18. A method for diagnosing carcinoma of the prostate, which comprises: (a) contacting a body fluid selected from the group consisting of blood, a blood fraction, and urine of a human being with immunoprecipitating antibodies to an antigen consisting essentially of the human prostate specific antigen of claim 1 associated with both normal and cancerous prostatic tissue which is distinct from prostatic acid phosphatase, said antibodies being free of cross-reactivity against prostatic acid phosphatases and against antigens associated with other carcinomas to form an immunoprecipitin complex between said antibodies and said antigen; and   (b) detecting the presence of said complex. .Iaddend. .Iadd.19. The method according to claim 18 in which the body fluid is blood. .Iaddend. .Iadd.20. The method according to claim 18 in which the body fluid is a blood fraction. .Iaddend. .Iadd.21. The method according to claim 18 in which the body fluid is serum. .Iaddend. .Iadd.22. The method according to claim 18 in which the body fluid is urine. .Iaddend. .Iadd.23. The method according to claim 18 in which the antibodies are monoclonal antibodies. .Iaddend. .Iadd.24. The method according to claim 18 wherein the complex is formed by countercurrent immunoelectrophoresis. .Iaddend. .Iadd.25. The method according to claim 18 wherein the complex is formed by immunologically reacting said antigen with antibodies thereto covalently   
     
     
        bound to a water-insoluble carrier. .Iaddend. .Iadd.26.  The method according to claim 18, wherein said complex is detected by 
     
     
        radioimmunoassay or by enzyme-linked immunoassay. .Iaddend. .Iadd.27.  The method according to claim 12 wherein the antigen is localized in a human 
     
     
        tissue sample. .Iaddend. .Iadd.28.  The method according to claim 33 in which the antibodies are monoclonal antibodies. .Iaddend. .Iadd.29. The method according to claim 33 wherein the complex is formed by countercurrent immunoelectrophoresis. .Iaddend. .Iadd.30. The method according to claim 33 wherein the complex is formed by immunologically reacting said antigen with antibodies thereto covalently bound to a water-insoluble carrier. .Iaddend. .Iadd.31. The method according to claim 33 wherein said complex is detected by radioimmunoassay or by enzyme-linked immunoassay. .Iaddend. .Iadd.32. The method according to claim 33 in which the tissue is prostatic tissue. .Iaddend.