Sandwich assay for small molecules

Abstract

Methods are disclosed for a sandwich assay for a small molecule having a molecular weight of about 500 to about 2,500. The method comprises the use of a first antibody that binds to the small molecule and 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 hapten that is not the small molecule or a derivative of the small molecule wherein the hapten comprises a moiety that is structurally similar to that of the second 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 determining a presence and/or amount of a small molecule having a molecular weight of about 500 to about 2,500 in a sample suspected of containing the small molecule, the method comprising:
 (a) providing in combination in a medium:
 (i) the sample, 
 (ii) a first binding partner that binds to and is specific for a first portion of the small molecule, and 
 (iii) a second binding partner that binds to the small molecule at a second portion of the small molecule other than the first portion, wherein the second binding partner is prepared from an immunogen that comprises a hapten that is not the small molecule or a derivative of the small molecule wherein the hapten comprises a moiety that is structurally similar to that of the second portion of the small molecule, 
   (b) incubating the medium under conditions for binding of the first binding partner and the second binding partner to the small molecule, and   (c) examining the medium for the presence of an immunocomplex comprising the small molecule, the first binding partner and the second binding partner, the presence and/or amount of the immunocomplex indicating the presence and/or amount of the small molecule in the sample.   
     
     
         2 . The method according to  claim 1 , wherein both the first binding partner and the second binding partner are antibodies. 
     
     
         3 . The method according to  claim 1 , wherein both the first binding partner and the second binding partner are monoclonal antibodies. 
     
     
         4 . The method according to  claim 1 , wherein the small molecule is selected from the group consisting of immunosuppressant drugs, cardiac glycosides, hormones and peptides. 
     
     
         5 . The method according to  claim 4 , wherein the small molecule is an immunosuppressant drug. 
     
     
         6 . The method according to  claim 5 , wherein the immunosuppressant drug is sirolimus or everolimus. 
     
     
         7 . The method according to  claim 6 , wherein the hapten of the immunogen is tacrolimus or a derivative thereof. 
     
     
         8 . The method according to  claim 1 , wherein one of the first binding partner and the second binding partner comprises a member of a signal producing system. 
     
     
         9 . The method according to  claim 1 , wherein one of the first binding partner and the second binding partner is bound to a support. 
     
     
         10 . A method of determining a presence and/or amount of an immunosuppressant drug in a sample suspected of containing the immunosuppressant drug, the method comprising:
 (a) providing in combination in a medium:
 (i) the sample, 
 (ii) a first antibody that binds to and is specific for a first portion of the immunosuppressant drug, and 
 (iii) a second antibody that binds to the immunosuppressant drug at a second portion of the immunosuppressant drug other than the first portion, wherein the second antibody is prepared from an immunogen that comprises a hapten that is not the immunosuppressant drug wherein the hapten comprises a moiety that is structurally similar to that of the second portion of the immunosuppressant drug, 
   (b) incubating the medium under conditions for binding of the first antibody and the second antibody to the immunosuppressant drug, and   (c) examining the medium for the presence of an immunocomplex comprising the immunosuppressant drug, the first antibody and the second antibody, the presence and/or amount of the immunocomplex indicating the presence and/or amount of the immunosuppressant drug in the sample.   
     
     
         11 . The method according to  claim 10 , wherein both the first antibody and the second antibody are monoclonal antibodies. 
     
     
         12 . The method according to  claim 10 , wherein the immunosuppressant drug is selected from the group consisting of sirolimus, everolimus, tacrolimus and cyclosporin. 
     
     
         13 . The method according to  claim 10 , wherein the immunosuppressant drug is sirolimus or everolimus and the hapten of the immunogen is tacrolimus or a derivative thereof. 
     
     
         14 . The method according to  claim 10 , wherein one of the first antibody and the second antibody comprises a member of a signal producing system. 
     
     
         15 . The method according to  claim 10 , wherein one of the first antibody and the second antibody is bound to a support. 
     
     
         16 . A method of determining a presence and/or amount of an immunosuppressant drug selected from the group consisting of sirolimus or everolimus in a sample suspected of containing the immunosuppressant drug, the method comprising:
 (a) providing in combination in a medium:
 (i) the sample, 
 (ii) a first monoclonal antibody that binds to and is specific for a first portion of the immunosuppressant drug, and 
 (iii) a second monoclonal antibody that binds to the immunosuppressant drug at a second portion of the immunosuppressant drug other than the first portion, wherein the second monoclonal antibody is prepared from an immunogen that comprises tacrolimus or a derivative of tacrolimus, 
   (b) incubating the medium under conditions for binding of the first monoclonal antibody and the second monoclonal antibody to the immunosuppressant drug, and   (c) examining the medium for the presence of an immunocomplex comprising the immunosuppressant drug, the first antibody and the second antibody, the presence and/or amount of the immunocomplex indicating the presence and/or amount of the immunosuppressant drug in the sample.   
     
     
         17 . The method according to  claim 16 , wherein one of the first monoclonal antibody and the second monoclonal antibody comprises a member of a signal producing system. 
     
     
         18 . The method according to  claim 16 , wherein one of the first monoclonal antibody and the second monoclonal antibody is bound to a support. 
     
     
         19 . The method according to  claim 16 , wherein the first portion consists essentially of a triene portion of the immunosuppressant drug. 
     
     
         20 . The method according to  claim 16 , wherein the first monoclonal antibody exhibits substantially no binding affinity for tacrolimus.