Novel complex of human kallikrein 2 (hK2) and protease inhibitor-6 (PI-6) in prostate tumor tissue and methods of using the complex

Abstract

The present invention provides a novel complex of hK2 and PI-6 and methods of using the novel complex. The novel complexes of hK2 and PI-6 of the present invention exist at an elevated level in prostate cancer tissues. Therefore, the hK2-PI6 complexes of the present invention may be used as a serum marker for detecting prostate cancer. They may also be used as an immunohistological marker to detect prostate cancer tissues. In accordance with the present invention, the hK2-PI6 complexes of the present invention may be detected in patient tissue samples by immunohistochemical and/or in patient fluid samples by in vitro immunoassay procedures. Diagnostic kits and diagnostic methods for detecting the existence of prostate cancer are also provided.

What is claimed is:  
     
         1 . An isolated and substantially pure complex consisting of protease inhibitor-6 (PI-6) and human kallikrein 2 (hK2).  
     
     
         2 . The complex of    claim 1   , wherein the complex consists of PI-6 and intact hK2, and the complex has a molecular weight of about 64 kDa measured by non-reducing polyacrylamide gel technique.  
     
     
         3 . The complex of    claim 1   , wherein the complex consists of PI-6 and a fragment of hK2, and the complex has a molecular weight of about 50 kDa measured by reducing polyacrylamide gel technique.  
     
     
         4 . The complex of    claim 3   , wherein the fragment of hK2 is a clipped hK2 clipped at arginine 145.  
     
     
         5 . The complex of    claim 1    being isolated from a prostate tissue.  
     
     
         6 . The complex of    claim 1    existing at an elevated level in a prostate tumor tissue sample.  
     
     
         7 . An antibody that is specifically immunoreactive with a complex consisting of protease inhibitor-6 (PI-6) and human kallikrein 2 (hK2).  
     
     
         8 . The antibody of    claim 7   , wherein the antibody is a polyclonal antibody.  
     
     
         9 . The antibody of    claim 7   , wherein the antibody is a monoclonal antibody.  
     
     
         10 . A method of detecting or determining in a sample a complex consisting of protease inhibitor-6 (PI-6) and human kallikrein 2 (hK2), the method comprising the steps of: 
 (a) contacting an amount of an agent which specifically binds to the hK2-PI6 complex to be detected with the sample under a condition that allows the formation of a ternary complex comprising the agent and the hK2-PI6 complex; and    (b) detecting or determining the presence or amount of the ternary complex.    
     
     
         11 . The method of    claim 10   , wherein the agent is an antibody.  
     
     
         12 . The method of    claim 11   , wherein the antibody is a monoclonal antibody.  
     
     
         13 . The method of    claim 10   , wherein the agent is an antibody and wherein the antibody is attached to a solid phase.  
     
     
         14 . The method of    claim 10   , wherein the agent is an antibody and wherein the antibody comprises a detectable label or binds to a detectable label to form a detectable complex.  
     
     
         15 . The method of    claim 10   , wherein the sample is a mammalian tissue sample.  
     
     
         16 . The method of    claim 15   , wherein the agent is an antibody.  
     
     
         17 . The method of    claim 15   , wherein in step (b), the complex is detected by a second agent which comprises a detectable label or which binds to a detectable label to form a detectable complex.  
     
     
         18 . The method of    claim 17   , wherein the second agent is an antibody.  
     
     
         19 . The method of    claim 15   , wherein the mammalian tissue is a human prostate tissue.  
     
     
         20 . The method of    claim 19   , wherein the human prostate tissue is a prostate cancer tissue.  
     
     
         21 . The method of    claim 10   , wherein the sample is a sample of human physiological fluid.  
     
     
         22 . The method of    claim 21   , wherein the human physiological fluid is selected from the group consisting of serum, seminal plasma, urine, and blood.  
     
     
         23 . An immunoassay for detecting hK2-PI6 complexes in a biological fluid sample comprising the steps of: 
 (a) providing a first antibody that recognizes one epitope of the hK2-PI6 complex, and a second antibody that recognizes another epitope of the hK2-PI6 complex,    (b) contacting the first antibody with the sample under a condition that allows the first antibody to bind to the complex to form a first complex that comprises the first antibody and the hK2-PI6 complex,    (c) contacting the second antibody with the first complex under a condition that allows the second antibody to bind to the hK2-PI6 complex to form a second complex that comprises the first antibody, the second antibody and the hK2-PI6 complex, and    (d) detecting and determining the second complex as a measurement of the hK2 PI6 complex contained in the sample.    
     
     
         24 . The immunoassay of    claim 23   , wherein the first and second antibodies are respectively an antibody that is specifically immunoreactive for the hK2-PI6 complex.  
     
     
         25 . The immunoassay of    claim 23   , wherein one of the first and second antibodies is an antibody against hK2, and another of the first and second antibodies is an antibody against PI-6.  
     
     
         26 . The immunoassay of    claim 23   , wherein one of the first and second antibodies is bound to a solid phase, and another of the first and second antibodies is labeled with a detectable label.  
     
     
         27 . A diagnostic kit for detecting or determining in a sample a complex consisting of PI-6 and hK2, the kit comprising a known amount of an agent which specifically binds to the complex, wherein the agent is detectably labeled or binds to a detectable label.  
     
     
         28 . The diagnostic kit of    claim 27   , wherein the sample is a sample of human physiological fluid.  
     
     
         29 . The diagnostic kit of    claim 27   , wherein the agent comprises an antibody that specifically binds to the complex.  
     
     
         30 . The diagnostic kit of    claim 29    further comprising a solid phase capable of having the antibody attached thereto.  
     
     
         31 . The diagnostic kit of    claim 27   , wherein the sample is a mammalian tissue sample.  
     
     
         32 . The diagnostic kit of    claim 31   , wherein the mammalian tissue sample is a human prostate tissue sample.  
     
     
         33 . The diagnostic kit of    claim 29   , wherein the antibody is a monoclonal antibody.  
     
     
         34 . A diagnostic kit for detecting or determining in a sample a complex consisting of PI-6 and hK2, the kit comprising a known amount of a first agent which specifically binds to the complex, wherein the first agent is detectably labeled or binds to a detectable label, and a known amount of a second agent which specifically binds to the complex, wherein the second agent is bound to a solid support.  
     
     
         35 . The kit of    claim 34   , wherein the first agent comprises an antibody that is specifically immunoreactive with the complex, and the second agent comprises an antibody that is specifically immunoreactive with the complex.  
     
     
         36 . The kit of    claim 34   , wherein one of the first and second agents comprises an antibody that is specifically immunoreactive with hK2, and the other of the first and second agents comprises an antibody that is specifically immunoreactive with PI-6.  
     
     
         37 . A diagnostic method for determining the presence or absence of prostate cancer comprising: 
 (a) contacting an amount of an agent, which specifically binds to a complex consisting of PI-6 and hK2, with a sample obtained from a human containing the complex under a condition sufficient to allow the formation of a ternary complex comprising the agent, the PI-6, and the hK2; and    (b) determining the amount of the ternary complex in the sample and correlating the amount of the ternary complex to the presence or absence of prostate cancer in the human.    
     
     
         38 . The diagnostic method of    claim 37   , wherein the sample is a sample of physiological fluid.  
     
     
         39 . The diagnostic method of    claim 37   , wherein the agent comprises an antibody.  
     
     
         40 . The diagnostic method of    claim 37   , wherein the sample is a mammalian tissue sample.  
     
     
         41 . The diagnostic method of    claim 40   , wherein the mammalian tissue sample is a prostate tissue sample.  
     
     
         42 . A diagnostic method for determining the presence or absence of prostate cancer comprising: 
 (a) contacting an amount of an agent, which specifically binds to PI-6, with a sample obtained from a human containing the PI-6 under a condition sufficient to allow the formation of a binary complex comprising the agent and the PI-6, wherein the sample is selected from a group consisting of a prostate tissue sample, serum, seminal plasma, urine and blood; and    (b) determining the amount of the complex in the sample and correlating the amount of the complex to the presence or absence of prostate cancer in the human.    
     
     
         43 . The diagnostic method of    claim 42   , wherein the sample is a prostate tissue sample.  
     
     
         44 . The diagnostic method of    claim 42   , wherein the agent comprises an antibody.  
     
     
         45 . A method for detecting and determining hK2-PI6 complexes in a tissue specimen, wherein the tissue specimen contains free hk2, free PI-6 and hK2-PI6 complex, the method comprising the steps of: 
 (a) providing a first antibody that is specific for the free hK2, a second antibody that is specific for the free PI-6, and a third antibody that recognizes and binds the hK2-PI6 complex;    (b) contacting the first and second antibodies with the tissue specimen under a condition that all the free hk2 and free PI-6 are blocked by the first and the second antibodies respectively;    (c) contacting the third antibody with the tissue specimen under a condition that allows the third antibody to bind the hK2-PI6 complex contained in the tissue specimen to form a ternary complex comprising the antibody and the hK2-PI6 complex; and    (d) detecting or determining the presence or amount of the ternary complex.    
     
     
         46 . The method of    claim 45   , wherein the third antibody is an antibody against hK2, which antibody recognizes and binds not only free hK2 but also the hK2-PI6 complex.  
     
     
         47 . The method of    claim 45   , wherein the third antibody may be an antibody against PI-6, which antibody recognizes and binds to not only free PI-6, but also the hK2-PI6 complex.