(T0930-01-07) Development and Validation of a Colorimetric Assay for Quantification of N-acetyl-β-D-glucosaminidase in Human Urine Using NAG-Based Calibration Standards and Quality Controls

Purpose: N-acetyl-β-D-glucosaminidase (NAG) is an intracellular lysosomal enzyme, present in the S3 segment of proximal tubular cells and distal nephron. Presence of NAG in the urine shows tubular damage and potentially kidney injury ^[1-2]. We do not use this assay for diagnostic purposes, but we evaluated commercial NAG quantification kits. These kits utilize the enzymatic product (pNP) as assay standards, which is sufficient for detecting the presence of NAG but doesn’t always lead to the most accurate results. To improve assay accuracy, we used NAG as the calibration standards and quality control samples (QCs). This demonstrated improved accuracy in NAG quantification. Additionally, physical adsorption was observed and caused loss of NAG activity in urine samples, further contributing to inaccurate quantification. To address this, two stabilizers were evaluated to prevent adsorption. Finally, assay accuracy and precision, sample stability, parallelism and selectivity were evaluated to ensure assay robust performance.

Methods: In commercial Abcam NAG kit, NAG uses a synthetic p-nitrophenol derivative (R-pNP) as a NAG substrate. Released pNP is measured at absorbance (OD 400 nm) and quantitated by pNP standards. The NAG activity is then calculated through the released pNP, reaction time, sample volume and dilution factor following the kit instruction. Urine samples with low enzyme activity require extended incubation time to yield detectable signals in assay. Using the activity calculation method in kit, NAG activity exhibited significant variability over reaction time in assay, which compromised the NAG quantification accuracy. For more accurate NAG quantification, Roche NAG control was used to prepare the NAG calibration standards (0.125 – 8.20 U/L) and QCs, and the substrates from Abcam NAG kit were used to react with NAG to release pNP which can be measured at OD 400 nm. The NAG activity was directly quantitated by the NAG calibration standard curve. To prevent adsorption, two of additives were evaluated - 5% BSA or 0.2% Tween®20. Matrix QCs with and without the additives were compared for recovery and adsorption. Assay accuracy and precision (A&P) assessment run included a set of 8 calibration standards, 3 replicates of buffer QC samples at the low, medium, and high concentration levels, and 3 replicates of endogenous QC samples at the endo low and high concentration levels. To assess the NAG stability in the urine sample we evaluated spiked samples with 0.2% Tween®20. We did three freeze-thaw cycles and 24-hour short-term stability of NAG. STS samples were maintained at ambient temperature under LED light conditions approximately 24 hours. To assess parallelism, 5 individual lots of Tween®20-treated human urine from individuals with suspected kidney injury were evaluated with and without MRD applied and diluted into range of the assay with a minimum dilution factor of 3. To confirm that spiked matrix selectivity did not compromise quantitation, Tween®20-treated human urine from 10 different donor lots were spiked with recombinant NAG at 2 concentrations based on the endogenous level determined for each donor lot.

Results: Following the Abcam NAG kit instruction, pNP-based standards demonstrated the inaccurate NAG quantification of NAG control samples prepared by Roche NAG control (Fig. 1A). To improve accuracy, NAG control was used to prepare both the calibration standards (STD range: 0.125-8.2 U/L) and QCs, which demonstrated accurate NAG quantification of QC samples (Fig. 1B). The assay with 3-hour reaction time demonstrated better QC accuracy (Fig. 1B). Additionally, pNP-based standards demonstrated the significant variation of NAG quantification of 16 individual urine lots in assay with different assay reaction times (0.5 and 3 hours) (Fig. 1C). However, NAG-based standards and QCs demonstrated comparable NAG quantification of 16 individual urine lots in assay with different assay reaction times (0.5 and 3 hours) Fig.1D). Taking these together, NAG-based standards and QCs improved accuracy in NAG quantification. In our study, spiking 0.2% Tween®20 or 5% BSA into samples both successfully prevented the adsorption in assay Fig. 2). However, 5% BSA spiking in samples significantly affected the NAG quantification (Fig. 2). 0.2% Tween®20 will be used as the stabilizer of urine sample to prevent the adsorption. To validate this optimized assay, assay accuracy and precision, sample stability, parallelism and selectivity were evaluated to ensure assay robust performance. Using NAG as the standards and QCs in combination with Abcam NAG kit, the colorimetric assay demonstrated great inter-run (Fig. 3A) and intra-run (Fig. 3B) accuracy and precision of QCs. 3FT and STS24 stability samples were within the acceptance criteria (Fig. 3C). For parallelism evaluation, the acceptable dilution range is DF 6.25 - 100 Total (DF 1 - 16 with MRD of 6.25) (Fig. 3D). For selectivity assessment, Tween®20-treated human urine samples spiked at low and high concentrations were within the acceptance criteria (Fig. 3E).

Conclusion: We developed and validated a colorimetric assay for accurate quantification of NAG in human urine, using NAG-based calibration standards and quality controls in combination with the commercial Abcam NAG kit.

References: 1. Minyoung Lee, Namki Hong, Yong-ho Lee, Eun Seok Kang, Bong Soo Cha, Byung-Wan Lee, Elevated N-acetyl-β-d-glucosaminidase, a urinary tubular damage marker, is a significant predictor of carotid artery atherosclerosis in type 1 diabetes, independent of albuminuria: A cross-sectional study, Journal of Diabetes and its Complications, 2018, 32, 8,777-783. https://doi.org/10.1016/j.jdiacomp.2018.05.019.
2. Kim SR, Lee YH, Lee SG, Kang ES, Cha BS, Kim JH, Lee BW. Urinary N-acetyl-β-D-glucosaminidase, an early marker of diabetic kidney disease, might reflect glucose excursion in patients with type 2 diabetes. Medicine (Baltimore). 2016 Jul;95(27): e4114. doi:10.1097/MD.0000000000004114.

Fig. 1 NAG-based standards and QCs improved accuracy in NAG quantification.

Fig. 2 0.2% Tween®20 successfully prevented the adsorption in assay

Fig. 3 Assay accuracy and precision, sample stability, parallelism and selectivity were evaluated to ensure assay robust performance.