Method for producing rapamycin-specific antibodies

Abstract

This invention relates to the production of polyclonal and monoclonal antibodies to specific sites of rapamycin (Sirolimus). The reactivity of these poly and monoclonal antibodies make them particularly useful for immunoassays for therapeutic drug monitoring (TDM). These immunoassays or TDM kits may include polyclonal or monoclonal antibodies to specific sites of rapamycin. These kits may also include various combinations of polyclonal antibodies, polyclonal and monoclonal antibodies or a panel of monoclonal antibodies. Rapamycin conjugate immunogens are prepared for the immunization of a host animal to produce antibodies directed against specific regions of the rapamycin molecule. By determining the specific binding region of particular antibody, immunoassays which are capable of distinguishing between the parent molecule, active metabolites, inactive metabolites and other structurally similar immunosuppressant compounds are developed. The use of divinyl sulfone (DVS) as the linker arm molecule for forming rapamycin-protein conjugate immunogens is described. DVS-linked rapamycin-protein conjugates were found to elicit antibodies with greater specificity to the rapamycin molecule than succinate linked conjugates.

1 . A method for producing an antibody to a rapamycin related compound selected from the group consisting of rapamycin, rapamycin metabolites, and rapamycin derivatives, said antibody raised against an immunogen comprising said rapamycin related compound conjugated to a protein carrier by a divinyl sulfone linker arm molecule, said method comprising: 
 a.) administering said immunogen to an animal so as to affect a specific antibody response to said rapamycin related compound;    b.) recovering an antibody to said rapamycin related compound from said animal; and    c.) determining that said antibody specifically recognizes a region of said rapamycin related compound, said region selected from the group consisting of the region from C1 to C8 and the region from C23 to C36.    
     
     
         2 . The method of  claim 1  wherein said protein carrier is selected from the group consisting of human serum albumin and keyhole limpet hemocyanin.  
     
     
         3 . The method of  claim 1  wherein said rapamycin related compound is conjugated at the C27 or at the C31 position to said protein carrier.  
     
     
         4 . The method of  claim 1  wherein said rapamycin metabolite is selected from the group consisting of M1, M3, M4, and M5.  
     
     
         5 . The method of  claim 1  wherein said rapamycin derivative is a 42-0-(2-hydroxy-ethyl) derivative of rapamycin.  
     
     
         6 . The method of  claim 5  wherein said 42-O-(2-hydroxy-ethyl) derivative is RAD.  
     
     
         7 . The method of  claim 1  wherein said rapamycin related compound is rapamycin.  
     
     
         8 . The method of  claim 1  wherein said immunogen is selected from the group consisting of Rapa-27-ox-DVS-KLH, Rapa-27-ox-DVS-HSA, Rapa-31-DVS-KLH, and Rapa-31-DVS-HSA.  
     
     
         9 . The method of  claim 1  wherein the step of recovering said antibody includes isolating at least one antibody-producing cell from said animal; immortalizing said antibody-producing cell to produce a hybridoma; and isolating said antibody from said hybridoma.  
     
     
         10 . The method of  claim 9  wherein said protein carrier is selected from the group consisting of human serum albumin and keyhole limpet hemocyanin.  
     
     
         11 . The method of  claim 9  wherein said rapamycin related compound is conjugated at the C27 or the C31 position to said protein carrier.  
     
     
         12 . The method of  claim 9  wherein said rapamycin metabolite is selected from the group consisting of M1, M3, M4, and M5.  
     
     
         13 . The method of  claim 9  wherein said rapamycin derivative is a 42-O-(2-hydroxy-ethyl) derivative of rapamycin.  
     
     
         14 . The method of  claim 13  wherein said 42-O-(2-hydroxy-ethyl) derivative is RAD.  
     
     
         15 . The method of  claim 9  wherein said rapamycin related compound is rapamycin.  
     
     
         16 . The method of  claim 11  wherein said immunogen is selected from the group consisting of Rapa-27-ox-DVS-KLH, Rapa-27-ox-DVS-HSA, Rapa-31-DVS-KLH, and Rapa-31-DVS-HSA.  
     
     
         17 . A method for producing at least one antibody to a rapamycin related compound selected from the group consisting of rapamycin and rapamycin metabolites, said at least one antibody raised against an immunogen comprising said rapamycin related compound conjugated to a protein carrier by a divinyl sulfone linker arm molecule, said method comprising: 
 a.) administering said immunogen to an animal so as to affect a specific antibody response to said rapamycin related compound;    b.) recovering an antibody to said rapamycin related compound from said animal;    c.) determining that said antibody specifically recognizes a region of said rapamycin related compound, said region selected from the group consisting of the region from C9 to C23, the region from C24 to C36, demethylated C7, demethylated C32, and demethylated C41.    
     
     
         18 . The method of  claim 17  wherein said protein carrier is selected from the group consisting of human serum albumin and keyhole limpet hemocyanin.  
     
     
         19 . The method of  claim 17  wherein said rapamycin related compound is conjugated at the C41 or at the C42 position to said protein carrier.  
     
     
         20 . The method of  claim 17  wherein said rapamycin metabolite is selected from the group consisting of M1, M2, M3, M4, and M5.  
     
     
         21 . The method of  claim 17  wherein said rapamycin related compound is rapamycin.  
     
     
         22 . The method of  claim 17  wherein said immunogen is selected from the group consisting of Rapa-42-DVS-KLH and Rapa-42-DVS-HSA.  
     
     
         23 . The method of  claim 17  wherein the step of recovering said antibody includes isolating at least one antibody-producing cell from said animal and immortalizing said antibody-producing cell to produce a hybridoma; and isolating said antibody from said hybridoma.  
     
     
         24 . The method of  claim 23  wherein said immunogen is selected from the group consisting of Rapa-42-DVS-KLH and Rapa-42-DVS-HSA.  
     
     
         25 . The method of  claim 23  wherein said protein carrier is selected from the group consisting of human serum albumin and keyhole limpet hemocyanin.  
     
     
         26 . The method of  claim 23  wherein said rapamycin related compound is conjugated at the C41 or at the C42 position to said protein carrier.  
     
     
         27 . The method of  claim 23  wherein said rapamycin metabolite is selected from the group consisting of M1, M2, M3, M4, and M5.  
     
     
         28 . The method of  claim 23  wherein said rapamycin related compound is rapamycin.