Sandwich assay design for small molecules

Abstract

Methods are disclosed of designing antibodies for a sandwich assay for a small molecule having a molecular weight of about 500 to about 2,000. The method comprises preparing a first antibody that binds to the small molecule, and preparing a second antibody that binds to the small molecule at a portion of the small molecule other than a portion to which the first antibody binds. The second antibody is prepared from an immunogen that comprises a predetermined portion of the small molecule. The antibodies may be employed in sandwich assays for the small molecule.

What is claimed is: 
     
         1 . A method of designing antibodies for a sandwich assay for a small molecule having a molecular weight of about 500 to about 2,000, the method comprising:
 (a) preparing a first antibody that binds to the small molecule, and   (b) preparing a second antibody that binds to the small molecule in a portion of the small molecule other than a portion to which the first antibody binds, wherein the second antibody is prepared from an immunogen that comprises a predetermined portion of the small molecule.   
     
     
         2 . The method according to  claim 1  wherein the first antibody is prepared from an immunogen that comprises a portion of the small molecule other than the predetermined portion. 
     
     
         3 . The method according to  claim 1  wherein the predetermined portion of the small molecule is obtained by modification of the small molecule to alter a spatial conformation of the small molecule. 
     
     
         4 . The method according to  claim 1  wherein the predetermined portion of the small molecule is a compound that consists essentially of the predetermined portion. 
     
     
         5 . The method according to  claim 1  wherein the small molecule is a macrolide. 
     
     
         6 . The method according to  claim 1  wherein the small molecule is an immunosuppressant drug. 
     
     
         7 . The method according to  claim 1  wherein one or both of the first antibody and the second antibody are monoclonal antibodies. 
     
     
         8 . A method of determining a presence and/or amount of a small molecule having a molecular weight of about 500 to about 2,000 in a sample suspected of containing the small molecule, the method comprising:
 (a) providing in combination in a medium:
 (i) the sample, 
 (ii) the first antibody for the small molecule according to  claim 1 , and 
 (iii) the second antibody for the small molecule according to  claim 1 , 
   (b) incubating the medium under conditions for binding of the first antibody and the second antibody to the small molecule, and   (c) examining the medium for the presence of an immunocomplex comprising the small molecule, the first antibody and the second antibody, the presence and/or amount of the immunocomplex indicating the presence and/or amount of the small molecule in the sample.   
     
     
         9 . The method according to  claim 8  wherein the small molecule is an immunosuppressant drug. 
     
     
         10 . A method of designing antibodies for a sandwich assay for a small molecule having a molecular weight of about 500 to about 2,000, the method comprising:
 (a) preparing a first monoclonal antibody that binds to a portion of the small molecule, and   (b) preparing a second monoclonal antibody that binds to the small molecule at a portion of the small molecule other than the portion to which the first monoclonal antibody binds, wherein the second monoclonal antibody is prepared from an immunogen that comprises the small molecule that is modified at the portion of the small molecule to which the first monoclonal antibody binds.   
     
     
         11 . The method according to  claim 10  wherein the first monoclonal antibody is prepared from an immunogen that comprises a portion of the small molecule other than the portion to which the second monoclonal antibody binds. 
     
     
         12 . The method according to  claim 10  wherein the derivatized portion of the small molecule alters a spatial conformation of the small molecule. 
     
     
         13 . The method according to  claim 10  wherein the small molecule is an immunosuppressant drug. 
     
     
         14 . The method according to  claim 13  wherein the immunosuppressant drug is sirolimus. 
     
     
         15 . The method according to  claim 14  wherein the derivatized portion of the sirolimus molecule comprises a triene. 
     
     
         16 . A method of determining a presence and/or amount of a small molecule having a molecular weight of about 500 to about 2,000 in a sample suspected of containing the small molecule, the method comprising:
 (a) providing in combination in a medium:
 (i) the sample, 
 (ii) the first monoclonal antibody for the small molecule according to  claim 10 , and 
 (iii) the second monoclonal antibody for the small molecule according to  claim 10 , 
   (b) incubating the medium under conditions for binding of the first monoclonal antibody and the second monoclonal antibody to the small molecule, and   (c) examining the medium for the presence of an immunocomplex comprising the small molecule, the first monoclonal antibody and the second monoclonal antibody, the presence and/or amount of the immunocomplex indicating the presence and/or amount of the small molecule in the sample.   
     
     
         17 . The method according to  claim 16  wherein the small molecule is an immunosuppressant drug. 
     
     
         18 . A method of designing antibodies for a sandwich assay for sirolimus, the method comprising:
 (a) preparing a first monoclonal antibody that binds to sirolimus, and   (b) preparing a second monoclonal antibody that binds to sirolimus in a portion of sirolimus other than the portion to which the first monoclonal antibody binds, wherein the second monoclonal antibody is prepared from an immunogen that is modified at the portion of sirolimus to which the first antibody binds.   
     
     
         19 . The method according to  claim 18  wherein the modified portion of sirolimus comprises a triene. 
     
     
         20 . A method of determining a presence and/or amount of a sirolimus in a sample suspected of containing sirolimus, the method comprising:
 (a) providing in combination in a medium:
 (i) the sample, 
 (ii) the first monoclonal antibody for sirolimus according to  claim 18 , and 
 (iii) the second monoclonal antibody for sirolimus according to  claim 18 , 
   (b) incubating the medium under conditions for binding of the first monoclonal antibody and the second monoclonal antibody to sirolimus, and   (c) examining the medium for the presence of an immunocomplex comprising sirolimus, the first monoclonal antibody and the second monoclonal antibody, the presence and/or amount of the immunocomplex indicating the presence and/or amount of sirolimus in the sample.