Doxorubicin immunoassay

Abstract

Novel conjugates of doxorubicin and novel doxorubicin immunogens derived from the 13 and 14 positions of doxorubicin and antibodies generated by these doxorubicin linked immunogens all of which are useful in immunoassays for the quantification and monitoring of doxorubicin in biological fluids.

1 . An antibody which binds selectively to doxorubicin and does not substantially bind to doxorubicin aglycone. 
     
     
         2 . The antibody of  claim 1 , wherein said antibody is derived from mice, sheep, rabbits or rats. 
     
     
         3 . The antibody of  claim 1 , wherein said antibody is a monoclonal antibody. 
     
     
         4 . The antibody of  claim 1 , wherein said antibody is derived from an immunogenic carrier having a reactive amino or thiol group polymer conjugated to a compound selected from the group consisting of compounds of the formula: 
       
         
           
           
               
               
           
         
         wherein A is 
       
       
         
           
           
               
               
           
         
         Y is an organic spacing group; 
         X is a functional group capable of binding to said carrier through said amino or thiol group; and 
         p is an integer from 0 to 1; 
       
       or compounds of the formula: 
       
         
           
           
               
               
           
         
         wherein X, Y and p are as above and B is —CH 2 — or 
       
       
         
           
           
               
               
           
         
       
       or mixtures thereof. 
     
     
         5 . The antibody of  claim 4 , wherein the compound conjugated to the immunogenic carrier for generating said antibody has the formula: 
       
         
           
           
               
               
           
         
         wherein p, X, Y and A are as above. 
       
     
     
         6 . The antibody of  claim 5 , wherein the carrier contains a thiol group and X in the compound which is conjugated to the immunogenic polymer is a functional group capable of reacting with said thiol. 
     
     
         7 . The antibody of  claim 6 , wherein X in said compound is 
       
         
           
           
               
               
           
         
       
     
     
         8 . The antibody of  claim 7 , wherein Y in said compound is lower alkyl. 
     
     
         9 . The antibody of  claim 8 , wherein the immunogenic carrier contains as the functional group: 
       
         
           
           
               
               
           
         
         wherein v is an integer from 1 to 6. 
       
     
     
         10 . The antibody of  claim 9 , wherein said antibody is derived from mice, sheep, rabbits or rats. 
     
     
         11 . A compound of the formula: 
       
         
           
           
               
               
           
         
         wherein A is 
       
       
         
           
           
               
               
           
         
         Y is an organic spacing group; 
         X is a functional group capable of binding to a thiol or amino group; and 
         p is an integer from 0 to 1. 
       
     
     
         12 . The compound of  claim 11 , wherein X is 
       
         
           
           
               
               
           
         
         wherein R 3  is hydrogen or taken together with its attached oxygen atom forms a reactive ester and R 4  is oxygen or sulfur. 
       
     
     
         13 . The compound of  claim 12 , wherein X is 
       
         
           
           
               
               
           
         
       
       and R 3  is hydrogen. 
     
     
         14 . The compound of  claim 12 , wherein X is 
       
         
           
           
               
               
           
         
       
       and R 3  forms a reactive ester. 
     
     
         15 . The compound of  claim 14 , wherein the ester formed is a lower alkyl ester, imidoester or amidoester. 
     
     
         16 . The compound of  claim 11 , wherein X is a functional group capable of binding to a thiol group. 
     
     
         17 . The compound of  claim 16 , wherein X is 
       
         
           
           
               
               
           
         
       
     
     
         18 . The compound of  claim 17 , wherein Y is alkylene containing from 1 to 10 carbon atoms. 
     
     
         19 . A compound of the formula: 
       
         
           
           
               
               
           
         
         wherein Y is an organic spacing group; 
         X is a functional group capable of binding to a thiol or an amino group; and 
         B is —CH 2 — or 
       
       
         
           
           
               
               
           
         
       
       or mixtures thereof. 
     
     
         20 . The compound of  claim 19 , wherein p is 0. 
     
     
         21 . The compound of  claim 20 , wherein X is 
       
         
           
           
               
               
           
         
         wherein R 3  is hydrogen or taken together with its attached oxygen atom forms a reactive ester and R 4  is oxygen or sulfur. 
       
     
     
         22 . The compound of  claim 21 , wherein X is 
       
         
           
           
               
               
           
         
       
       and R 3  is hydrogen. 
     
     
         23 . The compound of  claim 22 , wherein X is 
       
         
           
           
               
               
           
         
       
       and R 3  forms a reactive ester. 
     
     
         24 . The compound of  claim 23 , wherein the ester formed is a lower alkyl ester, imidoester or amidoester. 
     
     
         25 . The compound of  claim 19 , wherein p is 1. 
     
     
         26 . The compound of  claim 25 , wherein X is 
       
         
           
           
               
               
           
         
         wherein R 3  is hydrogen or taken together with its attached oxygen atom forms a reactive ester and R 4  is oxygen or sulfur. 
       
     
     
         27 . The compound of  claim 26 , wherein Y is alkylene containing from 1 to 10 carbon atoms, 
       
         
           
           
               
               
           
         
       
       wherein n and o are integers from 0 to 6, and m is an integer from 1 to 6. 
     
     
         28 . A conjugate of a carrier containing a functional thiol or amino group with a compound of the formula: 
       
         
           
           
               
               
           
         
         wherein A is 
       
       
         
           
           
               
               
           
         
         Y is an organic spacing group; 
         X is a functional group capable of binding to said thiol or amino group; and 
         p is an integer from 0 to 1. 
       
     
     
         29 . The conjugate of  claim 28 , wherein p is 0. 
     
     
         30 . The conjugate of  claim 29 , wherein X is 
       
         
           
           
               
               
           
         
       
       wherein R 3  is hydrogen or taken together with its attached oxygen atom forms a reactive ester and R 4  is oxygen or sulfur. 
     
     
         31 . The conjugate of  claim 28 , wherein p is 1 and Y is alkylene containing from 1 to 10 carbon atoms, 
       
         
           
           
               
               
           
         
         wherein n and o are integers from 0 to 6, and m is an integer from 1 to 6. 
       
     
     
         32 . The conjugate of  claim 31 , wherein said carrier has a reactive functional thiol group and X is a functional group capable of binding to said thiol group. 
     
     
         33 . The conjugate of  claim 32 , wherein X is 
       
         
           
           
               
               
           
         
       
     
     
         34 . The conjugate of  claim 33 , wherein Y is lower alkylene. 
     
     
         35 . The conjugate of  claim 34 , wherein said carrier contains a polymeric polyamine containing a functional group of the formula: 
       
         
           
           
               
               
           
         
         wherein v is an integer from 1 to 6. 
       
     
     
         36 . A conjugate of a carrier having a thiol or amine group with a compound of the formula: 
       
         
           
           
               
               
           
         
         wherein B is —CH 2 — or 
       
       
         
           
           
               
               
           
         
         Y is an organic spacing group; 
         X is a functional group capable of binding to said thiol or amino group in said carrier; and 
         p is an integer from 0 to 1. 
       
     
     
         37 . The conjugate of  claim 36 , wherein p is 0. 
     
     
         38 . The conjugate of  claim 36 , wherein p is 1. 
     
     
         39 . The conjugate of  claim 38 , wherein Y is alkylene containing from 1 to 10 carbon atoms, 
       
         
           
           
               
               
           
         
         wherein n and o are integers from 0 to 6, and m is an integer from 1 to 6. 
       
     
     
         40 . The conjugate of  claim 39 , wherein the carrier contains an immunogenic polymeric polymer containing more amino groups linked by 
       
         
           
           
               
               
           
         
         wherein R 4  is oxygen or sulfur. 
       
     
     
         41 . A kit for determining the presence of doxorubicin in a patient sample comprising reagents in separate containers, one of the reagents being a conjugate of a carrier containing a functional amino or thiol group with a compound selected from the groups consisting of compounds of the formula: 
       
         
           
           
               
               
           
         
         wherein A is 
       
       
         
           
           
               
               
           
         
         Y is an organic spacing group; 
         X is a functional group capable of binding to the amino or thiol group in said carrier; 
         p is an integer from 0 to 1; and 
         or a 14-substituted doxorubicin of the formula: 
       
       
         
           
           
               
               
           
         
         
           wherein X, Y and p are as above and B is —CH 2 — or 
         
       
       
         
           
           
               
               
           
         
       
       or mixtures thereof; and the second container containing an antibody substantially selectively reactive with doxorubicin and not substantially cross-reactive to doxorubicin aglycone. 
     
     
         42 . The kit of  claim 41 , wherein said conjugate is present in a predetermined amount in said first container. 
     
     
         43 . The kit of  claim 42 , wherein said kit is used to determine the amount of doxorubicin in said sample. 
     
     
         44 . The kit of  claim 41 , wherein, said antibody is generated from a conjugate of immunogenic carrier having a terminal functional amino or thiol group linked to said 13-substituted or 14-substituted doxorubicin. 
     
     
         45 . The kit of  claim 44 , wherein said conjugate is said 13-substituted doxorubicin. 
     
     
         46 . The kit of  claim 45 , wherein said carrier has a reactive terminal functional thiol group and X is a terminal functional group capable of binding to said thiol group. 
     
     
         47 . The kit of  claim 46 , wherein X is