Assays for detection of phenylalanine ammonia-lyase and antibodies to phenylalanine ammonia-lyase

Abstract

Provided herein are methods of detecting the presence of a pegylated enzyme, an enzyme-specific antibody (e.g., a neutralizing antibody or of a particular isotype), or a polyethylene glycol (PEG)-specific antibody in a sample, such as a bodily fluid or tissue of a patient. In certain embodiments, the enzyme is phenylalanine ammonia-lyase (PAL), such as Anabaena variabilis (Av) PAL administered to the patient as part of an enzyme substitution therapy for diseases or disorders, such as phenylketonuria (PKU), or cancer therapy.

1 . A method of detecting the presence of a pegylated phenylalanine ammonia-lyase (PAL-PEG) in a sample, comprising:
 (a) acidifying the PAL-PEG in the sample by adding an acidification reagent;   (b) neutralizing the acidified PAL-PEG in the sample by adding a neutralization buffer;   (c) contacting the sample with an immobilized first antibody that immunospecifically binds to phenylalanine ammonia-lyase (PAL);   (d) optionally removing unbound sample;   (e) contacting the sample bound to the immobilized first antibody with a detectable second antibody, wherein the second antibody immunospecifically binds to PEG;   (f) optionally removing unbound second antibody; and   (g) detecting the presence of the second antibody bound to the sample;   
       wherein the PAL-PEG is a pegylated recombinant  Anabaena variabilis  phenylalanine ammonia-lyase (rAvPAL-PEG) or variant thereof, and 
       wherein detection above background of an amount of the second antibody bound to the sample indicates the presence of PAL-PEG in the sample. 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . The method according to  claim 1 , further comprising measuring the amount of the PAL-PEG in the sample. 
     
     
         5 - 11 . (canceled) 
     
     
         12 . The method according to  claim 1 , wherein the rAvPAL-PEG variant is rAvPAL-PEG_C503S, rAvPAL-PEG_C565S or rAvPAL-PEG_C565SC503S. 
     
     
         13 . (canceled) 
     
     
         14 . The method according to  claim 1 , wherein the sample comprises a bodily fluid or tissue. 
     
     
         15 . The method of  claim 14 , wherein the bodily fluid is blood, serum or plasma. 
     
     
         16 . The method according to  claim 1 , wherein the sample is from a patient. 
     
     
         17 . The method of  claim 16 , wherein the patient is a mammal. 
     
     
         18 . The method of  claim 17 , wherein the mammal is a human. 
     
     
         19 . The method according to  claim 18 , wherein the patient is a patient having elevated phenylalanine levels. 
     
     
         20 . The method according to  claim 19 , wherein the patient has phenylketonuria. 
     
     
         21 . The method according to  claim 18 , wherein the patient has cancer. 
     
     
         22 . The method according to  claim 18 , wherein the patient has been, is, or will be administered a rAvPAL-PEG or variant thereof. 
     
     
         23 . The method according to  claim 22 , wherein the rAvPAL-PEG variant is rAvPAL-PEG_C503S, rAvPAL-PEG_C565S or rAvPAL-PEG_C565SC503S. 
     
     
         24 . A kit comprising:
 (a) an acidification reagent for acidifying a PAL-PEG, wherein the PAL-PEG is a rAvPAL-PEG or variant thereof;   (b) a neutralization buffer for neutralizing the acidified PAL-PEG;   (c) a first antibody that immunospecifically binds to PAL that is optionally immobilized on a solid support; and   (d) a detectable second antibody that immunospecifically binds to PEG.   
     
     
         25 - 31 . (canceled) 
     
     
         32 . The kit according to  claim 24 , wherein one or more components of the kit are in a one or more containers, and wherein the kit further comprises instructions for use. 
     
     
         33 - 37 . (canceled) 
     
     
         38 . The kit according to  claim 24 , wherein the rAvPAL-PEG variant is rAvPAL-PEG_C503S, rAvPAL-PEG_C565S or rAvPAL-PEG_C565SC503S. 
     
     
         39 . (canceled) 
     
     
         40 . The method according to  claim 1 , wherein the acidification reagent is 0.1 M glycine, pH 2.7. 
     
     
         41 . The method according to  claim 1 , wherein the neutralization buffer is 0.5 M Tris-HCl, pH 8.5. 
     
     
         42 . The kit according to  claim 24 , wherein the acidification reagent is 0.1 M glycine, pH 2.7. 
     
     
         43 . The kit according to  claim 24 , wherein the neutralization buffer is 0.5 M Tris-HCl, pH 8.5.