Validation of Aspartylglucosaminidase Activity Assay for Human Serum Samples: Establishment of a Biomarker for Diagnostics and Clinical Studies

Abstract

Novel treatment strategies are emerging for rare, genetic diseases, resulting in clinical trials that require adequate biomarkers for the assessment of the treatment effect. For enzyme defects, biomarkers that can be assessed from patient serum, such as enzyme activity, are highly useful, but the activity assays need to be properly validated to ensure a precise, quantitative measurement. Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by the deficiency of the lysosomal hydrolase aspartylglucosaminidase (AGA). We have here established and validated a fluorometric AGA activity assay for human serum samples from healthy donors and AGU patients. We show that the validated AGA activity assay is suitable for the assessment of AGA activity in the serum of healthy donors and AGU patients, and it can be used for diagnostics of AGU and, potentially, for following a treatment effect.

Keywords: aspartylglucosaminuria; biomarkers; enzymes; lysosomal storage disorders; protein glycosylation.

Figure 1

Linearity of the AMC standard curve. AMC-standards were prepared as outlined in Section 4.4. Raw AMC fluorescence (in artificial units) was plotted against the corresponding AMC amount in pmol. The slope, y-intercept, and R-square were calculated by linear regression analysis. Symbols show the mean ± SD of five independent analyses.

Figure 2

Accuracy of AMC detection. Inter-assay accuracy was determined by spiking of samples with defined amounts of AMC. (a) artificial serum matrix; (b) AGU patient serum, or (c) serum of healthy donors. The samples were supplemented with 10–100 pmol of AMC and incubated for 24 h to mimic the AGA activity assay protocol. Nominal and measured AMC values were plotted against each other (black symbols, left axis). R-square was calculated by linear regression analysis, and recovery rate (blue symbols, right axis) was computed from the determined value and expressed as % from the true value. The values represent the mean ± SD of three independent experiments.

Figure 3

Within-run precision. AGA activity in three technical replicates with 10 µL serum from 6 healthy donors or 20 µL of serum from 6 AGU patients was measured following the validated AGA activity assay protocol. Data show one representative run out of at least 3 runs.

Figure 4

Between-run precision. (a) Between-run precision of the AGA activity assay was assessed with serum samples of 8 healthy donors. Each symbol represents one independent measurement out of at least 4 experiments. (b) Between-run precision of the AGA activity assay in AGU patient sera. Samples from 20 AGU patients were assessed by three independent experiments. The data for two healthy donors from panel (a) are shown for comparison.

Figure 5

Dilution integrity. AGA activity was measured with different amounts of serum (1–10 µL) from healthy donors. All samples were diluted with NaCl or artificial serum to 20 µL total volume for the assay. AGA activity in the serum samples of six healthy donors was measured according to the validated protocol. (a) The mean values (pmol AMC) of the six healthy donors were used for linear regression analysis. (b) The mean AMC values back-calculated as pmol AMC per liter over the sample volume range; (c) Individual AGA activities (mU/L) in the serum samples of the six healthy donors.

Figure 6

Schematic presentation of the steps of the AGA activity assay. See Section 4.4 for details.