Immunoassays for everolimus

Abstract

Immunoassays for the detection of everolimus are provided. Compounds for producing antibodies for everolimus, as well as antibodies produced therefrom, are also provided.

1 . A competitive immunoassay to determine the presence of everolimus in a sample comprising 
 an antibody capable of specifically binding everolimus, and    an everolimus compound conjugated to a detectable label,    wherein the conjugated everolimus compound is configured to compete with the everolimus in the sample to bind with the antibody, and    wherein the label provides a signal indicative of a concentration of everolimus in the sample when the everolimus in the sample is present in therapeutic drug monitoring concentrations.    
     
     
         2 . The competitive immunoassay of  claim 1  wherein the range of therapeutic drug concentrations includes everolimus from about 3 to about 15 ng/ml.  
     
     
         3 . The competitive immunoassay of  claim 1  wherein the signal is indicative of the concentration of everolimus when everolimus is present from about 0 to about 40 ng/ml.  
     
     
         4 . The competitive immunoassay of  claim 1  wherein the antibody was produced using an antigen having the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 n is 0 or 1;  
 X is a linker chain comprising 3-10 carbon or hetero atoms, wherein the linker chain may be substituted or unsubstituted and may be straight or branched;  
 Y is selected from the group consisting of —C(O)—, —NH—, —S—, —CH 2 — and —O—; and  
 Z is an antigenic carrier.  
 
     
     
         5 . The competitive immunoassay of  claim 4  wherein the antibody was produced using an antigen having the formula:  
       
         
           
           
               
               
           
         
       
     
     
         6 . The competitive immunoassay of  claim 1  wherein the assay is selected from the group consisting of an FPIA, an homogeneous microparticle (immunoturbidimetric) immunoassay, a cloned enzyme donor immunoassay (CEDIA), and a chemiluminescent heterogeneous immunoassay, and lateral flow immunoassay.  
     
     
         7 . The competitive immunoassay of  claim 1  wherein the conjugated everolimus compound is a compound of the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 X 1  is a linker chain comprising one or more atoms, each of which may be substituted or unsubstituted and may be branched or unbranched;  
 Y is selected from the group consisting of —C(O)—, —NH—, —S—, —CH 2 — and —O—; and  
 Z is the label.  
 
     
     
         8 . The competitive immunoassay of  claim 7  wherein 
 X 1  is —O—CH 2 —, and    Y is —C(O)—.    
     
     
         9 . The competitive immunoassay of  claim 1  wherein the antibody exhibits strong binding and strong inhibition of greater than 50% displacement over an assay range with everolimus.  
     
     
         10 . The competitive immunoassay of  claim 1  provided as a kit.  
     
     
         11 . The competitive immunoassay of  claim 10 , further comprising a calibrator.  
     
     
         12 . A method for determining the amount of everolimus in a sample comprising 
 mixing the sample with 
 an antibody capable of specifically binding everolimus, and  
 an everolimus compound conjugated to a detectable label,  
   wherein the conjugated everolimus compound is configured to compete with the everolimus in the sample to bind with the antibody,    measuring a signal from the detectable label indicative of a concentration of everolimus in the sample, and determining the amount of everolimus in the sample.    
     
     
         13 . The method of  claim 14  wherein the everolimus in the sample is present in therapeutic drug monitoring concentrations.  
     
     
         14 . The method of  claim 15  wherein the range of therapeutic drug concentrations includes everolimus from about 3 to about 15 ng/ml.  
     
     
         15 . The method of  claim 13  wherein the antibody was produced using an antigen having the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 n is 0 or 1;  
 X is a linker chain comprising 3-10 carbon or hetero atoms, wherein the linker chain may be substituted or unsubstituted and may be straight or branched;  
 Y is selected from the group consisting of —C(O)—, —NH—, —S—, —CH 2 — and —O—; and  
 Z is an antigenic carrier.  
 
     
     
         16 . The method of  claim 13  wherein the sample is a body fluid.  
     
     
         17 . The method of  claim 13  wherein the mixing step comprises first mixing the antibody and the everolimus compound conjugated to the detectable label and then adding the sample.  
     
     
         18 . The method of  claim 13  wherein the mixing step comprises first mixing the antibody and the sample and then adding the everolimus compound conjugated to the detectable label.  
     
     
         19 . A compound having the following structure:  
       
         
           
           
               
               
           
         
       
       wherein 
 n is 0 or 1;  
 X is a linker chain comprising 3-10 carbon or hetero atoms, wherein the linker chain may be substituted or unsubstituted and may be straight or branched;  
 Y is selected from the group consisting of —C(O)—, —NH—, —S—, —CH 2 — and —O—; and  
 Z is an antigenic carrier or a label.  
 
     
     
         20 . The compound of  claim 19  wherein 
 n=1 and    X is a linker group comprising 4-6 substituted or unsubstituted straight or branched chain carbon or hetero atoms.    
     
     
         21 . The compound of  claim 20  wherein 
 X is —CH 2 —CH 2 -CH 2 —CH 2 -CH 2 — and    Y is —C(O)—.    
     
     
         22 . The compound of  claim 21  wherein Z is the antigenic carrier.  
     
     
         23 . An antibody produced using the compound of 22 that is capable of specific binding to everolimus.  
     
     
         24 . The antibody of  claim 23  wherein the antibody is a monoclonal antibody.  
     
     
         25 . The antibody of  claim 23  wherein the antibody is a polyclonal antibody:  
     
     
         26 . An immunoassay kit for detecting everolimus in a sample comprising 
 the antibody of  claim 23 , and    a detectable label,    wherein the label is configured to produce a detectable signal when the sample, the antibody, and the label are mixed together and everolimus is present in the sample.    
     
     
         27 . The immunoassay of  claim 26  wherein the label is linked to the antibody or to a secondary antibody having specificity for the antibody.  
     
     
         28 . The immunoassay of  claim 26  wherein the label is linked to an everolimus molecule that is configured to compete with everolimus in the sample for binding with the antibody.