Gemcitabine immunoassay

Abstract

The present invention comprises novel conjugates and immunogens derived from gemcitabine and unique antibodies generated by using gemcitabine linked immunogens, which conjugates immunogens and antibodies, are useful in immunoassays for the quantification and monitoring of gemcitabine in biological fluids.

1 . An immunoassay for detecting gemcitabine in a sample comprising providing a mixture of a) said sample, b) an antibody selectively reactive with gemcitabine and not substantially cross-reactive with 2′,2′-difluoro-2′-deoxyuridine and tetrahydrouridine, and c) a conjugate of a carrier having either a reactive thiol or amino 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 carrier through said amino or thiol group; and 
         p is an integer from 0 to 1 
       
       or salts thereof; 
       causing the gemcitibine in the sample and said conjugate in said mixture to bind with said antibody and thereafter measuring the amount of said conjugate in said mixture which is bound or unbound to said antibody whereby the presence of gemcitibine in the sample can be determined. 
     
     
         2 . The process of  claim 1 , wherein the sample is a human sample. 
     
     
         3 . The immunoassay of  claim 2 , wherein said antibody is generated from an immunogen comprising an immunogenic carrier having a reactive thiol or amino group conjugated to a compound of the formula: 
       
         
           
           
               
               
           
         
         wherein p, X, Y and B are as above; 
       
       or salts thereof.
 wherein p, X, Y and A are as above. 
 
     
     
         4 . The immunoassay of  claim 3 , wherein the carrier contains a thiol group and X is the compound which is linked to the immunogenic polymer is a functional group capable of reacting with said thiol. 
     
     
         5 . The immunoassay of  claim 4 , wherein X is 
       
         
           
           
               
               
           
         
       
     
     
         6 . The immunoassay of  claim 5 , wherein Y is lower alkylene. 
     
     
         7 . The immunoassay of  claim 6  wherein the immunogenic carrier contains as the functional group 
       
         
           
           
               
               
           
         
         wherein v is an integer from 1 to 6. 
       
     
     
         8 . The immunoassay of  claim 2 , wherein the antibody is attached to a solid support. 
     
     
         9 . The immunoassay of  claim 8 , wherein the solid support is microtiter plates. 
     
     
         10 . The immunoassay of  claim 9 , wherein the solid support is nanoparticles. 
     
     
         11 . An antibody which binds selectively to gemcitibine and does not have any substantially cross reactivity to 2′,2′-difluoro-2′-deoxyuridine and tetrahydrouridine. 
     
     
         12 . The antibody of  claim 11  wherein said antibody has a cross-reactivity with regard to 2′,2′-difluoro-2′-deoxyuridine and tetrahydrouridine of less than 20%, based upon said antibody's reactivity with gemcitibine. 
     
     
         13 . The antibody of  claim 12  wherein said cross reactivity is less than 10% 
     
     
         14 . The antibody of  claim 11 , wherein said antibody is derived from mice, sheep, rabbits or rats. 
     
     
         15 . The antibody of  claim 14 , wherein said antibody is a monoclonal antibody. 
     
     
         16 . The antibody of  claim 11 , 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 B is —CH 2 — or 
       
       
         
           
           
               
               
           
         
         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 salts thereof. 
     
     
         17 . The antibody of  claim 16 , 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. 
     
     
         18 . The antibody of  claim 17 , wherein X in said compound is 
       
         
           
           
               
               
           
         
       
     
     
         19 . The antibody of  claim 18 , wherein Y in said compound is lower alkylene. 
     
     
         20 . The antibody of  claim 19 , wherein the immunogenic carrier contains as the functional group: 
       
         
           
           
               
               
           
         
         Wherein v is an integer from 1 to 6. 
       
     
     
         21 . The antibody of  claim 20 , wherein said antibody is derived from mice, sheep, rabbits or rats. 
     
     
         22 . 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 carrier through said amino or thiol group; and 
         p is an integer from 0 to 1 
         X is a functional group capable of binding to a thiol or an amino group; 
         and B is —CH 2 — or 
       
       or salts thereof. 
     
     
         23 . The compound of  claim 22 , wherein p is 0. 
     
     
         24 . The compound of  claim 21 , 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. 
       
     
     
         25 . The compound of  claim 24 , wherein X is 
       
         
           
           
               
               
           
         
       
       and R 3  is hydrogen. 
     
     
         26 . The compound of  claim 24 , wherein X if 
       
         
           
           
               
               
           
         
       
       and OR 3  forms a reactive ester. 
     
     
         27 . The compound of  claim 26 , wherein the ester formed is a lower alkyl ester, imidoester or amidoester. 
     
     
         28 . The compound of  claim 22 , wherein p is 1. 
     
     
         29 . The compound of  claim 28 , 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. 
       
     
     
         30 . The compound of  claim 28 , wherein Y is alkylene containing from 1 to 10 carbon atoms, 
       
         
           
           
               
               
           
         
         wherein n and o are integers from 0 to 6, and in is an integer from 1 to 6. 
       
     
     
         31 . 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 carrier through said amino or thiol group; and 
         p is an integer from 0 to 1 
       
       or salts thereof: 
     
     
         32 . The conjugate of  claim 31 , wherein p is 0. 
     
     
         33 . The conjugate of  claim 32 , wherein p is 1. 
     
     
         34 . The conjugate of  claim 33 , 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. 
       
     
     
         35 . The conjugate of  claim 34 , wherein the carrier contains an immunogenic polymeric polymer containing one or more amino groups linked by 
       
         
           
           
               
               
           
         
         wherein R 4  is oxygen or sulfur. 
       
     
     
         36 . A kit for determining the presence of gemcitibine 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 B is —CH 2 — or 
       
       
         
           
           
               
               
           
         
         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 salts thereof; and the second container containing an antibody substantially selectively reactive with gemcitibine and not substantially cross-reactive to 2′,2′-difluoro-2′-deoxyuridine and tetrahydrouridine 
     
     
         37 . The kit of  claim 36 , wherein said conjugate is present in a predetermined amount in said first container. 
     
     
         38 . The kit of  claim 37 , wherein said kit is used to determine the amount of gemcitibine in said sample. 
     
     
         39 . The kit of  claim 38 , wherein said carrier has a reactive terminal functional thiol group and X is a terminal functional group capable of binding to said thiol group. 
     
     
         40 . The kit of  claim 39 , wherein X is