ASSAYS FOR DETECTION OF PHENYLALANINE AMMONIA-LYASE AND ANTIBODIES TO PHENYLALANINE AMMONIA-LYASE
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.