Immunoassay kit for measuring cyclosporine, its metabolites and derivatives

Abstract

This invention relates to the production of polyclonal and monoclonal antibodies to specific regions of cyclosporine (CSA) and/or CSA metabolites/derivatives. The reactivity of these polyclonal 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 CSA and/or CSA metabolites. These kits may also include various combinations of polyclonal antibodies, polyclonal and monoclonal antibodies or a panel of monoclonal antibodies. Cyclosporine or CSA metabolite conjugate immunogens are prepared for the immunization of a host animal to produce antibodies directed against specific regions of the CSA or CSA metabolite molecule. By determining the specific binding region of a 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 cyclosporine and cyclosporine metabolite protein conjugate immunogens is described.

1 - 59 . (canceled)  
     
     
         60 . An immunoassay kit for measuring the amount of a cyclosporine related compound in a sample, said cyclosporine related compound selected from the group consisting of CSA, a CSA metabolite, and a CSA derivative, said kit comprising an antibody selected from the group consisting of: 
 CSA-2G9 produced by the hydridoma cell line deposited with the ATCC as PTA-4170;    AM1-7F5 produced by the hydridoma cell line deposited with the ATCC as PTA-4142;    AM1-3B1 produced by the hydridoma cell line deposited with the ATCC as PTA-4154;    AM1-2E10 produced by the hydridoma cell line deposited with the ATCC as PTA-4141;    AM9-1-3C1 produced by the hydridoma cell line deposited with the ATCC as PTA-4155;    AM19-1-5D2 produced by the hydridoma cell line deposited with the ATCC as PTA-4163;    AM19-1-5B3 produced by the hydridoma cell line deposited with the ATCC as PTA-4147;    AM19-9-5A6 produced by the hydridoma cell line deposited with the ATCC as PTA-4150;    AM9-1-6D4 produced by the hydridoma cell line deposited with the ATCC as PTA-4157;    AM9-1-7D2 produced by the hydridoma cell line deposited with the ATCC as PTA-4144;    AM9-9-11G9 produced by the hydridoma cell line deposited with the ATCC as PTA-4158;    AM9-9-6C3 produced by the hydridoma cell line deposited with the ATCC as PTA-4146;    AM19-1-7E12 produced by the hydridoma cell line deposited with the ATCC as PTA-4161;    AM9-1-2A11 produced by the hydridoma cell line deposited with the ATCC as PTA-4143;    AM19-9-1E11 produced by the hydridoma cell line deposited with the ATCC as PTA-4149;    AM19-9-1D8 produced by the hydridoma cell line deposited with the ATCC as PTA-4148;    AM19-9-2G9 produced by the hydridoma cell line deposited with the ATCC as PTA-4160;    AM9-9-11H11 produced by the hydridoma cell line deposited with the ATCC as PTA-4159;    AM9-9-4F5 produced by the hydridoma cell line deposited with the ATCC as PTA-4145; and    AM9-1-4D6 produced by the hydridoma cell line deposited with the ATCC as PTA-4156.    
     
     
         61 . The kit of  claim 60  wherein said antibody is selective for said CSA metabolite and said metabolite is selected from the group consisting of AM1, AM1c, AM9, and AM19.  
     
     
         62 . The kit of  claim 60  wherein said antibody is selective for CSA.  
     
     
         63 . The kit of  claim 60  wherein said antibody is selective for said cyclosporine derivative and said derivative is modified at the amino acid 1 residue.  
     
     
         64 . The kit of  claim 60  wherein said antibody is selected from the group consisting of AM1-2E10, AM19-9-5A6, and AM9-1-6D4.  
     
     
         65 . The kit of  claim 60  wherein said antibody is selected from the group consisting of CSA-2G9, AM1-2E10, AM1-7F5, and AM19-1-7E12.  
     
     
         66 . The kit of  claim 60  wherein said antibody is CSA-2G9.  
     
     
         67 . The kit of  claim 60  wherein said antibody is selected from the group consisting of AM9-1-7D2, AM9-9-6C3 and AM9-9-11G9.  
     
     
         68 . An immunoassay kit for measuring the amount of a cyclosporine related compound in a sample, said cyclosporine related compound selected from the group consisting of CSA, a CSA metabolite, and a CSA derivative, said kit comprising a first antibody and a second antibody, said first antibody and said second antibody independently selected from the group consisting of: 
 CSA-2G9 produced by the hydridoma cell line deposited with the ATCC as PTA-4170;    AM1-7F5 produced by the hydridoma cell line deposited with the ATCC as PTA-4142;    AM1-3B1 produced by the hydridoma cell line deposited with the ATCC as PTA-4154;    AM1-2E10 produced by the hydridoma cell line deposited with the ATCC as PTA-4141;    AM9-1-3C1 produced by the hydridoma cell line deposited with the ATCC as PTA-4155;    AM19-1-5D2 produced by the hydridoma cell line deposited with the ATCC as PTA-4163;    AM19-1-5B3 produced by the hydridoma cell line deposited with the ATCC as PTA-4147;    AM19-9-5A6 produced by the hydridoma cell line deposited with the ATCC as PTA-4150;    AM9-1-6D4 produced by the hydridoma cell line deposited with the ATCC as PTA-4157;    AM9-1-7D2 produced by the hydridoma cell line deposited with the ATCC as PTA-4144;    AM9-9-11G9 produced by the hydridoma cell line deposited with the ATCC as PTA-4158;    AM9-9-6C3 produced by the hydridoma cell line deposited with the ATCC as PTA-4146;    AM19-1-7E12 produced by the hydridoma cell line deposited with the ATCC as PTA-4161;    AM9-1-2A11 produced by the hydridoma cell line deposited with the ATCC as PTA-4143;    AM19-9-1E 11 produced by the hydridoma cell line deposited with the ATCC as PTA-4149;    AM19-9-1D8 produced by the hydridoma cell line deposited with the ATCC as PTA-4148;    AM19-9-2G9 produced by the hydridoma cell line deposited with the ATCC as PTA-4160;    AM9-9-11H11 produced by the hydridoma cell line deposited with the ATCC as PTA-4159;    AM9-9-4F5 produced by the hydridoma cell line deposited with the ATCC as PTA-4145; and    AM9-1-4D6 produced by the hydridoma cell line deposited with the ATCC as PTA-4156.    
     
     
         69 . The kit of  claim 68  wherein said first antibody is specific for at least one CSA metabolite selected from the group consisting of AM1, AM1c, AM9, and AM19.  
     
     
         70 . The kit of  claim 69  wherein said second antibody is selective for CSA.  
     
     
         71 . The kit of  claim 68  wherein said first antibody is purified.  
     
     
         72 . The kit of  claim 71  wherein said first antibody is selected from the group consisting of: 
 AM1-2E10 produced by the hydridoma cell line deposited with the ATCC as PTA-4141;  
 AM19-9-5A6 produced by the hydridoma cell line deposited with the ATCC as PTA-4150;  
 AM9-1-6D4 produced by the hydridoma cell line deposited with the ATCC as PTA-4157;  
 AM9-1-7D2 produced by the hydridoma cell line deposited with the ATCC as PTA-4144;  
 AM9-9-11G9 produced by the hydridoma cell line deposited with the ATCC as PTA-4158;  
 AM9-9-6C3 produced by the hydridoma cell line deposited with the ATCC as PTA-4146;  
 AM19-1-7E12 produced by the hydridoma cell line deposited with the ATCC as PTA-4161;  
 AM9-1-2A11 produced by the hydridoma cell line deposited with the ATCC as PTA-4143;  
 AM19-9-1E11 produced by the hydridoma cell line deposited with the ATCC as PTA-4149;  
 AM19-9-1D8 produced by the hydridoma cell line deposited with the ATCC as PTA-4148;  
 AM19-9-2G9 produced by the hydridoma cell line deposited with the ATCC as PTA-4160; and  
 AM9-1-4D6 produced by the hydridoma cell line deposited with the ATCC as PTA-4156.  
 
     
     
         73 . The kit of  claim 71  wherein said second antibody is selected from the group consisting of CSA-2G9, AM1-2E10, AM1-7F5, and AM19-1-7E12.  
     
     
         74 . The kit of  claim 68  wherein said compound is modified at the amino acid 1 residue and said second antibody is selected from the group consisting of CSA-2G9, AM1-2E10, AM1-7F5, and AM19-1-7E1  
     
     
         75 . The kit of  claim 68  wherein said compound is CSG and said second antibody is CSA-2G9.  
     
     
         76 . The kit of  claim 72  wherein said second antibody is purified and is selected from the group consisting of: 
 AM1-2E10 produced by the hydridoma cell line deposited with the ATCC as PTA-4141;  
 AM19-9-5A6 produced by the hydridoma cell line deposited with the ATCC as PTA-4150;  
 AM9-1-6D4 produced by the hydridoma cell line deposited with the ATCC as PTA-4157;  
 AM19-1-7E12 produced by the hydridoma cell line deposited with the ATCC as PTA-4161;  
 AM9-1-2A11 produced by the hydridoma cell line deposited with the ATCC as PTA-4143;  
 AM19-9-1E11 produced by the hydridoma cell line deposited with the ATCC as PTA-4149;  
 AM19-9-1D8 produced by the hydridoma cell line deposited with the ATCC as PTA-4148;  
 AM19-9-2G9 produced by the hydridoma cell line deposited with the ATCC as PTA-4160; and  
 AM9-1-4D6 produced by the hydridoma cell line deposited with the ATCC as PTA-4156.  
 
     
     
         77 . The kit of  claim 76  further comprising a third antibody, said third antibody selected from the group consisting of CSA-2G9, AM1-2E10, and AM1-7F5.  
     
     
         78 . The kit of  claim 68  further comprising a third antibody, said third antibody selected from the group consisting of CSA-2G9, AM1-2E10, and AM1-7F5.  
     
     
         79 . The kit for measuring AM9 in a biological sample, said kit comprising at least one antibody selected from the group consisting of AM9-1-7D2, AM9-911G9, and AM9-9-6C3 and at least one antibody selected from the group consisting of CSA-2G9, AM1-2E10, AM19-7E12, and AM1-7F5.  
     
     
         80 . The kit of  claim 68  wherein said sample is a biological sample.  
     
     
         81 . The kit of  claim 68  wherein said first antibody binds to one face of the CSA molecule and said second antibody binds to the other face of the CSA molecule.  
     
     
         82 . A kit for quantifying CSA in an automated TDM, said kit comprising an antibody selected from the group consisting of: 
 CSA-2G9 produced by the hydridoma cell line deposited with the ATCC as PTA-4170;    AM1-7F5 produced by the hydridoma cell line deposited with the ATCC as PTA-4142;    AM1-3B1 produced by the hydridoma cell line deposited with the ATCC as PTA-4154;    AM1-2E10 produced by the hydridoma cell line deposited with the ATCC as PTA-4141;    AM9-1-3C1 produced by the hydridoma cell line deposited with the ATCC as PTA-4155;    AM19-1-5D2 produced by the hydridoma cell line deposited with the ATCC as PTA-4163;