Methods for detection of cyclosporin a

Abstract

Methods and reagents are disclosed for determining the presence and/or amount of cyclosporin A in a medium suspected of containing cyclosporin A. In the method a combination is provided in a medium. The combination comprises (i) the sample, (ii) a first member of a signal producing system (sps) associated with a first support wherein the first sps member is capable of activating a second member of the sps and wherein the first support is associated with a first member of a specific binding pair, and (iii) the second sps member associated with a second support wherein the second sps member is activatable by the first sps member. The second support comprises either (I) cyclosporin C or cyclosporin A and the combination further comprises a conjugate of an antibody for cyclosporin A and a second member of the specific binding pair or (II) antibody for cyclosporin A and the combination further comprises a conjugate of cyclosporin A and a second member of the specific binding pair. The combination is subjected to conditions for binding of cyclosporin A to the antibody for cyclosporin A. The first sps member is activated and the amount of signal generated by the second sps member is detected. The amount of signal is related to the presence and/or amount of cyclosporin A in the sample.

1 . A method for determining the presence and/or amount of cyclosporin A in a medium suspected of containing cyclosporin A, the method comprising:
 (a) providing in combination in a medium:
 (i) the sample, 
 (ii) a first member of a signal producing system (sps) associated with a first support wherein the first sps member is capable of activating a second member of the sps and wherein the first support is associated with a first member of a specific binding pair, and 
 (iii) the second sps member associated with a second support wherein the second sps member is activatable by the first sps member and wherein the second support comprises
 (I) cyclosporin C or cyclosporin A and the combination further comprises a conjugate of an antibody for cyclosporin A and a second member of the specific binding pair or 
 (II) antibody for cyclosporin A and the combination further comprises a conjugate of cyclosporin A and a second member of the specific binding pair, 
 
   (b) subjecting the combination to conditions for binding of cyclosporin A to the antibody for cyclosporin A, and   (c) activating the first sps member and detecting the amount of signal generated by the second sps member, the amount of signal being related to the presence and/or amount of cyclosporin A in the sample.   
   
   
       2 . The method according to  claim 1  wherein the specific binding pair comprises biotin and a biotin-binding partner. 
   
   
       3 . The method according to  claim 2  wherein the first member of the specific binding pair is streptavidin. 
   
   
       4 . The method according to  claim 1  wherein the second support comprises (I). 
   
   
       5 . The method according to  claim 1  wherein the first sps member is a sensitizer. 
   
   
       6 . The method according to  claim 5  wherein the second sps member is a chemiluminescent composition. 
   
   
       7 . A method for determining the presence and/or amount of cyclosporin A in a medium suspected of containing cyclosporin A, the method comprising:
 (a) providing in combination in a medium:
 (i) the sample, 
 (ii) a photosensitizer associated with a first particle and being capable of generating singlet oxygen wherein the first particle comprises a biotin binding partner, 
 (iii) a conjugate of cyclosporin A and biotin, and 
 (iv) a chemiluminescent composition activatable by singlet oxygen and associated with a second particle wherein the second particle comprises an antibody for cyclosporin A, 
   (b) subjecting the combination to conditions for binding of cyclosporin A to the antibody for cyclosporin A, and   (c) irradiating the photosensitizer with light and detecting the amount of luminescence generated by the chemiluminescent composition, the amount of luminescence being related to the presence and/or amount of cyclosporin A in the sample.   
   
   
       8 . The method according to  claim 7  wherein the biotin-binding partner is streptavidin. 
   
   
       9 . The method according to  claim 7  wherein the biotin is bis-biotin. 
   
   
       10 . The method according to  claim 7  wherein the second particle is a polystyrene particle having the chemiluminescent composition incorporated therein. 
   
   
       11 . A method for determining the presence and/or amount of cyclosporin A in a medium suspected of containing cyclosporin A, the method comprising:
 (a) providing in combination in a medium:
 (i) the sample, 
 (ii) a photosensitizer associated with a first particle and being capable of generating singlet oxygen wherein the first particle comprises a biotin binding partner, 
 (iii) a chemiluminescent composition activatable by the singlet oxygen and associated with a second particle wherein the second particle comprises cyclosporin C and 
 (iv) a conjugate of an antibody for cyclosporin A and biotin, 
   (b) subjecting the combination to conditions for binding of cyclosporin A, if present, to the antibody for cyclosporin A, and   (c) irradiating the photosensitizer with light and detecting the amount of luminescence generated by the chemiluminescent composition, the presence and/or amount of luminescence being related to the amount of cyclosporin A in the sample.   
   
   
       12 . The method according to  claim 11  wherein the biotin-binding partner is streptavidin. 
   
   
       13 . The method according to  claim 11  wherein the biotin is bis-biotin. 
   
   
       14 . The method according to  claim 11  wherein the second particle is a polystyrene particle having the chemiluminescent composition incorporated therein. 
   
   
       15 . A composition comprising cyclosporin A conjugated, at an amide nitrogen at position 7 or position 8, to bis-biotin by means of a linking group that has the formula:
   —(CH 2 ) e OC(O)NH(CH 2 ) f C(O)NH((CH 2 ) 2 O) g (CH 2 ) h NHC(O)D   
     wherein D is 
     
       
         
         
             
             
         
       
     
     wherein G is —C(O)(CH 2 ) j NH—, and 
     wherein e is 1 to 3,f is 1 to 3 μg is 1 to 4,h is 1 to 3, and j is 1 to 6. 
   
   
       16 . The composition according to  claim 15  wherein e is 2, f is 1, g is 2, h is 2 and j is 5.