Monoclonal antibodies to tacrolimus and immunoassay methods for tacrolimus

Abstract

An IgG 1 λ monoclonal antibody to the immunosuppressive drug tacrolimus has improved properties. In particular, this monoclonal antibody, designated 1H6, has reduced cross-reactivity to several tacrolimus metabolites. This antibody is suitable for performance of immunoassays such as homogeneous immunoassays to detect or determine the presence or concentration of tacrolimus in samples such as blood samples. The invention further includes derivatives of tacrolimus derivatized at a non-binding portion of the molecule useful in immunizing antibody-producing animals and in producing such monoclonal antibodies, as well as labeled derivatives of tacrolimus useful as tacrolimus analogues in such assays. The invention further includes immunoassay methods for the detection of tacrolimus and test kits useful in performing such immunoassays.

1 . (canceled)  
   
   
       2 . A monoclonal antibody to tacrolimus that: 
 (a) competes with the IgG 1 λ monoclonal antibody designated 1H6 at least about 80% as effectively on a molar basis as compared with the IgG 1 λ monoclonal antibody designated 1H6 as measured by competition assays; and    (b) has less than about 10% cross-reactivity with each of 15-demethyl tacrolimus, 31-demethyl tacrolimus, 13,31-didemethyl tacrolimus, 15,31-didemethyl tacrolimus, and 12-hydroxy tacrolimus.    
   
   
       3 . The monoclonal antibody of  claim 2  wherein the antibody competes at least about 90% as effectively on a molar basis as the monoclonal antibody designated 1H6 and has less than about 8% cross-reactivity with each of 15-demethyl tacrolimus, 31-demethyl tacrolimus, 13,31-didemethyl tacrolimus, 15,31-didemethyl tacrolimus and 12-hydroxy tacrolimus.  
   
   
       4 . (canceled)  
   
   
       5 . A hybridoma producing the monoclonal antibody of  claim 2 .  
   
   
       6 . The monoclonal antibody of  claim 2  wherein at least some of the constant regions of the antibody are replaced by human constant regions so that the monoclonal antibody is humanized.  
   
   
       7 . The monoclonal antibody of  claim 6  wherein the antibody competes at least about 90% as effectively on a molar basis as compared with the IgG 1 λ monoclonal antibody designated 1H6 and wherein the antibody has less than about 8% cross-reactivity with each of 15-demethyl tacrolimus, 31-demethyl tacrolimus, 13,31-didemethyl tacrolimus, 15,31-didemethyl tacrolimus, and 12-hydroxy tacrolimus.  
   
   
       8 - 18 . (canceled)  
   
   
       19 . A conjugate comprising the antibody of  claim 2  conjugated directly or indirectly to a detectable label.  
   
   
       20 . The conjugate of  claim 19  wherein the detectable label is selected from the group consisting of an enzyme label, a radioactive label, a fluorescent label, a chemiluminescent label, a bioluminescent label, and a particulate label.  
   
   
       21 . The conjugate of  claim 20  wherein the label is an enzyme label.  
   
   
       22 . A conjugate comprising the antibody of  claim 6  conjugated directly or indirectly to a detectable label.  
   
   
       23 . The conjugate of  claim 22  wherein the detectable label is selected from the group consisting of an enzyme label, a radioactive label, a fluorescent label, a chemiluminescent label, a bioluminescent label, and a particulate label.  
   
   
       24 . The conjugate of  claim 23  wherein the label is an enzyme label.  
   
   
       25 - 41 . (canceled)  
   
   
       42 . A method of detecting or determining tacrolimus comprising the steps of: 
 (a) providing a sample suspected of containing tacrolimus;    (b) reacting the sample with: 
 (i) the antibody of  claim 2;  and  
 (ii) optionally, a tacrolimus analogue; wherein one of the antibody or the tacrolimus analogue is labeled with a label producing a detectable signal; and  
   (c) observing or measuring one of: 
 (i) the signal associated with tacrolimus bound to antibody;  
 (ii) the signal associated with tacrolimus unbound to antibody; or  
 (iii) the total signal present;  
 in order to detect or determine the presence or concentration of tacrolimus in the sample.  
   
   
   
       43 . The method of  claim 42  wherein the sample is reacted with a tacrolimus analogue labeled with an enzyme label and the total signal present is observed or measured to detect or determine the presence or concentration of tacrolimus in the sample.  
   
   
       44 - 56 . (canceled)  
   
   
       57 . A test kit comprising, packaged in separate containers: 
 (a) the antibody of  claim 2;  and    (b) a tacrolimus analogue labeled directly or indirectly with an enzyme label.    
   
   
       58 - 82 . (canceled)