ENZYMATIC ASSAYS FOR QUANTIFYING THERAPY IN SUBJECTS WITH MUCOPOLYSACCHARIDOSIS TYPE I OR II

1 . A system for measuring the levels and/or activity of iduronate-2-sulfatase (IDS) in a biological sample, the system comprising the following separate reaction mixtures:

(a) three or more separate reference standard reactions comprising a detectably-labeled IDS substrate comprising 4-methylumbelliferone-alpha-L-idopayranosiduronic Acid 2-Sufate Disodium salt (4MU-IDS), and recombinant IDS (rIDS), wherein the three or more reference standard reactions include different concentrations of rIDS;

(b) at least first, second and third separate quality control reactions comprising 4MU-IDS and rIDS, wherein the first quality control reaction comprises rIDS at a low quality control level (LQC), the second quality control reaction comprises rIDS at a medium quality control level (MQC) and the third quality control reaction comprises rIDS at a high quality control level (HQC)

(c) three or more separate substrate reactions comprising different concentrations of the detectably-labeled substrate; and

(d) a plurality of sample reactions comprising the biological sample and the detectably-labeled IDS substrate.

2 . The system of claim 1 , comprising duplicate reactions of at least the reference standards and quality control reactions.

3 . The system of claim 1 , wherein the at least first, second and third separate quality control reactions further comprise additional comprising quality control reactions with rIDS at the lower and/or upper levels of quantification and wherein the separate reaction mixtures of the system are included on the same matrix.

4 . The system of claim 3 , wherein for the three or substrate reactions comprise 4MU concentrations of 0.235 μM to 50 μM and further wherein the concentration of 4MU in the reference standard reactions comprise serial dilutions of a 1.25 to 2.5 mM stock 4MU solution.

5 . A method of measuring the levels and/or activity of IDS in a biological sample, the method comprising the steps of:

(a) providing the system of separate reaction mixtures of claim 1 ;

(b) incubating the reactions;

(c) stopping the reactions of step (b) after a period of time;

(d) adding recombinant iduronidase (rIDUA) to each of the separate reactions;

(e) incubating the reactions of step (d);

(f) measuring the levels of detectable label from each reaction;

(g) generating (i) a reference standard curve from the levels of detectable label measured in the reference standard reactions and (ii) a substrate standard curve from the levels of detectable label measured in the substrate reactions;

(h) determining and/or quantifying the level and/or activity of IDS in the biological sample by measuring the levels of detectable label in the sample reactions and comparing the detected sample levels with the reference and substrate standard curves to determine enzyme activity in the sample.

6 . The method of claim 7 , further comprising determining an acceptable level criteria for the sample reaction measurements using one or more of the following parameters:

calculating the concentration of the standards, wherein at least 75% of the calculated concentrations for the standards must have a relative error (RE) within ±20% of low quality control (LQC), medium quality control (MQC) and high quality control (HQC);

calculating the concentration of the standards, wherein at least 75% of the calculated concentrations for the standards must have an RE within ±25% of the lower limit of quantification (LLOQ) or upper limit of quantification ULOQ;

substrate concentrations having a TE of ≤30% for LQC, MQC, HQC or ULOQ;

substrate concentrations having a TE of ≤40% for LLOQ;

% CVs of blank-corrected RFU for the reference and substrate standards is equal to or less than 20%; and/or

the substrate and/or reference curves have r 2 >0.98.

7 . The method of claim 5 , wherein the IDS standard curve as described herein providing the enzyme activity covers the range of quantification from at least 0.78 to 167 nmol/hr/mL.

8 . The method of claim 5 , wherein the sample is a plasma sample, a leukocyte sample, or a blood sample obtained from an MPS II subject.

9 . The method of claim 8 , wherein the MPS II subject has been treated with ERT and/or gene therapy reagents.

10 . The method of claim 5 , wherein the reactions of step (b) are incubated for 1-3 hours and the reactions of step (d) are incubated for 1 to 24 hours, further wherein the reactions are incubated at physiological temperature.

11 . The method of claim 5 , wherein the samples are contained in a micro plate and the levels of the detectable label are measured using a micro plate reader.

12 . A system for measuring the levels and/or activity of IDUA in a biological sample, the system comprising the following separate reaction mixtures:

(a) three or more separate reference IDUA reactions comprising a detectably-labeled IDUA substrate comprising 4-methylumbelliferone-alpha-L-iduronide (4MU-IDUA) and recombinant IDS (rIDUA), wherein the three or more reference standard reactions include different concentrations of rIDUA;

(b) three or more separate substrate reactions comprising the detectably-labeled IDUA substrate

(c) at least first, second and third separate quality control reactions comprising 4MU-IDUA and rIDUA, wherein the first quality control reaction comprises rIDUA at a low quality control level, the second quality control reaction comprises rIDUA at a mid quality control level and the third quality control reaction comprises rIDUA at a high quality control level; and

(d) a plurality of sample reactions comprising the biological sample and the detectably-labeled IDUA substrate.

13 . The system of claim 12 , comprising duplicate reactions of at least the reference standards and quality control reactions.

14 . The system of claim 12 , the at least first, second and third separate quality control reactions further comprise additional comprising quality control reactions with rIDUA at the lower and/or upper levels of quantification and wherein the separate reaction mixtures of the system are included on the same matrix.

15 . The system of claim 14 , wherein for the three or substrate reactions comprise 4MU concentrations of 0.235 μM to 50 μM and further wherein the concentration of 4MU in the reference standard reactions comprise serial dilutions of a 1.25 to 2.5 mM stock 4MU solution.

16 . A method of measuring the levels and/or activity of IDUA in a biological sample, the method comprising the steps of:

(a) providing the system of separate reaction mixtures of claim 12 ;

(b) incubating the reactions;

(c) measuring the levels of detectable label from each reaction;

(d) generating (i) a reference standard curve from the levels of detectable label measured in the reference standard reactions and (ii) a substrate standard curve from the levels of detectable label measured in the substrate reactions;

(e) determining and/or quantifying the level and/or activity of IDUA in the biological sample by measuring the levels of detectable label in the sample reactions and comparing the detected sample levels with the reference and substrate standard curves to determine enzyme activity in the sample.

17 . The method of claim 16 , further comprising determining an acceptable level criteria for the sample reaction measurements using one or more of the following parameters:

calculating the concentration of the standards, wherein at least 75% of the calculated concentrations for the standards must have an RE within ±20% of LQC, MQC and HQC;

calculating the concentration of the standards, wherein at least 75% of the calculated concentrations for the standards must have an RE within ±25% of the LLOQ or ULOQ;

substrate concentrations having a TE of ≤30% for LQC, MQC, HQC or ULOQ;

substrate concentrations having a TE of ≤40% for LLOQ;

% CVs of blank-corrected RFU for the reference and substrate standards is equal to or less than 20%; and/or

the substrate and/or reference curves have r 2 >0.98.

18 . The method of claim 16 , wherein the IDUA standard curve as described herein providing the enzyme activity covers the range of quantification from at least 0.66 to 167 nmol/hr/mL.

19 . The method of claim 16 , wherein the sample is a plasma sample, a leukocyte sample or a blood sample obtained from an MPS I subject.

20 . The method of claim 19 , wherein the MPS I subject has been treated with ERT and/or gene therapy reagents.

21 . The method of claim 16 , wherein the reactions of step (b) are incubated for 1-3 hours, further wherein the reactions are incubated at physiological temperature.

22 . The method of claim 16 , wherein the samples are contained in a micro plate and the levels of the detectable label are measured using a micro plate reader.