A technology and reagent for detecting NAG by VRA-NAG substrate method

Abstract

The application belongs to the field of biological detection, and particularly relates to a technology and a reagent for detecting NAG by a VRA-NAG substrate method. The linear range of the NAG detection reagent provided by the application can reach (0, 500] U / L; within the range, when the anti-acid is less than or equal to 340 micromoles / L, urea is less than or equal to 42.9 millimoles / L, uric acid is less than or equal to 1.4 millimoles / L, albumin is less than or equal to 100 milligrams / dL, bilirubin is less than or equal to 342 micromoles / L, lactic acid is less than or equal to 6.6 millimoles / L, glucose is less than or equal to 55 millimoles / L, triglyceride is less than or equal to 37 millimoles / L, and oxalic acid is less than or equal to 81 micromoles / L, these components have no obvious interference on the test. Moreover, the relative standard deviation caused by the accelerated aging of the detection reagent for 10 days is not more than ±10%. Compared with the prior art, the kit provided by the application has high sensitivity, wide linear range, strong specificity and good stability, and is conducive to early detection and early treatment of kidney diseases in clinical practice.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, and in particular relates to a technique and reagent for detecting NAG using the VRA-NAG (5-[4-(3-methoxy-phenylene-raphanin)]-3-acetamide-N-acetamido-β-D-glucosidase) substrate method. Background Technology

[0002] NAG (non-glucosidase) is an important lysosomal hydrolase in the human body, with a relative molecular mass of 130–140 kDa and a half-life of only 5 minutes in plasma. NAG is widely present in the human body; when kidney function is impaired, lysosomes rupture, releasing NAG and causing an increase in NAG levels. NAG testing can very sensitively reflect kidney damage. Since the glomeruli cannot filter NAG from plasma, in recent years, the level of NGA in urine has become a more ideal indicator for monitoring kidney disease, and the detection of urinary NGA levels has clinical applicability.

[0003] There are several existing NAG detection technologies, mainly including fluorescence spectrophotometry and ultraviolet spectrophotometry. Among them, fluorescence spectrophotometry requires expensive instruments and substrate solutions, making it unsuitable as a routine detection method in general hospitals.

[0004] Driven by demand, many rapid and inexpensive ultraviolet spectrophotometric detection methods have been developed, such as the CNP-NAG method, PNP-NAG method, MNP-G1CNAc method, and VRA-NAG method. The principle is that NAG catalyzes the decomposition of the substrate. By measuring the absorbance of the substrate or product at a specific wavelength at different times, the concentration change can be determined, thus indicating the concentration of the catalytic enzyme NAG. The CNP-NAG substrate method uses a substrate that dissolves slowly and has poor stability; it requires heating to 60℃ for 6 hours to completely dissolve, and after dissolution, it can only be stored at 4℃ for 7 days, making it inconvenient to use. The decomposition products of the PNP-NAG method are very close to the color of urine, which can easily cause serious interference with detection. The maximum absorption peak of the MNP-G1CNAc substrate method is at 500nm, and it is also easily affected by the slightly yellow color of urine. Currently, the linear range of existing products on the market is relatively narrow, generally only (0, 200] U / L, which needs to be improved. In summary, the interference of other components on photodetection, reagent instability, and reagent aging are problems that need to be solved in the development of NAG ultraviolet spectrophotometric detection reagents.

[0005] In recent years, the development and use of VRA-NAG substrates have improved substrate instability and reduced interference from reaction substrates or products on photodetection to some extent. However, the problem of interference from other components during detection still needs to be addressed, and the stability of reagents and the sensitivity of detection need to be further improved. Therefore, it is necessary to further optimize the detection of NAG. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a NAG detection reagent and detection method with good stability and high sensitivity.

[0007] This invention provides an NAG detection kit, which includes reagent R1; the reagent R1 includes: reaction substrate VRA-NAG, ascorbic acid oxidase, surfactant, buffer solution, protectant and stabilizer; wherein the stabilizer includes magnesium chloride, EDTA, AES and HPD.

[0008] In some embodiments, without the addition of AES or HPD, the anti-aging ability is not as good as that of R1 provided by the present invention.

[0009] In the reagent R1: the buffer solution includes a citric acid buffer solution; the surfactant includes one or more of Triton X-100, Tween-20, and PEG 6000; and the protectant includes one or more of sucrose, trehalose, and glucose.

[0010] Specifically, the stabilizer includes: 0.5–50 mmol / L magnesium chloride, 0.5–50 mmol / L EDTA, 0.1%–7.5% (V / V) AES and 0.1%–7.5% (V / V) HPD.

[0011] Specifically, in reagent R1: the concentration of the reaction substrate VRA-NAG is 0.5–50 mmol, the concentration of the ascorbic acid oxidase is 1–5 KU / L, the concentration of the surfactant is 0.02%–0.1% (V / V), the buffer solution is 50–200 mmol / L, the pH of the buffer solution is 4.6–5.0, and the concentration of the protective agent is 0.5–25 g / L.

[0012] Preferably, reagent R1 comprises: 15 mmol / L VRA-NAG, 2 KU / L ascorbic acid oxidase, 10 mmol / L magnesium chloride, 2 mmol / L EDTA, 1% (V / V) AES, 2.5% (V / V) HPD, 100 mmol / L citrate buffer solution at pH 4.8, 0.1% Triton X-100 (volume concentration), and 5 g / L trehalose protectant.

[0013] The reagent R1 also includes a preservative, which includes NaN3 or Proclin 300.

[0014] Specifically, in the R1 reagent, the concentration of the preservative is 0.02% to 0.1% (V / V).

[0015] The detection kit provided by the present invention further includes reagent R2, which comprises: sodium carbonate buffer solution, surfactant and preservative, wherein the surfactant comprises one or more of Triton X-100, Tween-20 and PEG 6000; and the preservative comprises NaN3 or Proclin 300.

[0016] Specifically, the reagent R2 comprises: a sodium carbonate buffer solution of 0–200 mmol / L with a pH of 10–10.8, 0.02%–0.1% (V / V) of surfactant, and 0.02%–0.1% (V / V) of preservative.

[0017] Preferably, the reagent R2 comprises: 100 mmol / L sodium carbonate buffer solution at pH 10.2, 0.1% (V / V) of Triton X-100 surfactant, and 0.1% (V / V) of Proclin 30.

[0018] This invention also provides a method for testing NAG content using the aforementioned kit: First, add 750 μL of reagent R1 and 50 μL of sample (S) to a cuvette, mix well, incubate at 37°C for 5 min, and read the absorbance A1. Then, add 250 μL of reagent R2, mix well, incubate at 37°C for 5 min, and read the absorbance A2. Finally, calculate ΔA (ΔA = A2 - A1).

[0019] The NAG detection kit provided by this invention has a linear detection range of (0, 500] U / L. Within this range, when antiserum ≤340 μmol / L, urea ≤42.9 mmol / L, uric acid ≤1.4 mmol / L, albumin ≤100 mg / dL, bilirubin ≤342 μmol / L, lactate ≤6.6 mmol / L, glucose ≤55 mmol / L, triglycerides ≤37 mmol / L, and oxalate ≤81 μmol / L, these components do not significantly interfere with the test. Furthermore, the relative standard deviation caused by accelerated aging of the detection kit for 10 days does not exceed ±10%. Compared with the prior art, the kit provided by this invention has high sensitivity, a wide linear range, strong specificity, and good stability, which is beneficial for the early detection and treatment of kidney diseases in clinical practice. Attached Figure Description

[0020] Figure 1 The linear fitting curve of the repeatability test is shown.

[0021] Figure 2 The image shows a comparison of linear fits within the linear range (0, 200 U / L) and the extended range (0, 300 U / L) as shown in the kit instructions. Detailed Implementation

[0022] This invention provides a NAG detection kit. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0023] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0024] The present invention will be further illustrated below with reference to the embodiments:

[0025] This method uses the 5-[4-(3-methoxy-phenylene-raphanin)]-3-acetamide-N-acetamido-β-D-glucosidase (VRA-NAG) substrate method to detect urinary NAG activity. VRA-NAG is a newly synthesized NAG substrate in recent years, with good water solubility and strong stability. Moreover, the concentration can reach five times the Km value of the substrate decomposition reaction catalyzed by NAG enzyme under the conditions of pH 4.75-5.0. At this substrate concentration, the competitive inhibition of the reaction by urea in urine has been eliminated.

[0026] The technology provided by this invention has high sensitivity, wide linear range, good precision, and high accuracy. It is simple and quick to operate and can be applied to the large-scale sample operation of fully automated biochemical analyzers. It is also suitable for small-scale sample testing of small fully automated biochemical analyzers, semi-automated biochemical analyzers, and other instruments, which is beneficial for the early detection and treatment of kidney diseases in clinical practice.

[0027] Example 1: VRA-NAG method for determining NAG content.

[0028] (1) Reagent preparation

[0029] Reagent R1:

[0030] Citric acid buffer solution: 100 mmol / L, pH 4.8;

[0031] VRA-NAG: 15 mmol / L

[0032] Ascorbic acid oxidase: 2 KU / L;

[0033] Surfactant: Triton X-100 0.1% (v / v);

[0034] Preservative: Potassium sorbate 0.1% (V / V);

[0035] Stabilizers: magnesium chloride 10 mmol / L, EDTA 2 mmol / L, 1% (V / V) AES and 2.5% (V / V) HPD;

[0036] Protectant: Trehalose 5g / L.

[0037] Reagent R2:

[0038] Sodium carbonate buffer solution: 100 mmol / L, pH 10.2;

[0039] Surfactant: Triton X-100 0.1% (v / v);

[0040] Preservative: Proclin 300 0.1% (V / V).

[0041] (2) Measurement conditions

[0042] Table 1 Experimental Measurement Conditions

[0043] dominant wavelength 505nm Sample volume (S) 50μL subwavelength 700nm Volume of reagent R1 750μL reaction temperature 37℃ Volume of reagent R2 250μL Cuvette optical path 1cm reaction type Endpoint method

[0044] (3) Operation steps

[0045] To test the absorbance of the samples under the conditions shown in Table 1, first add 750 μL of reagent R1 and 50 μL of sample (S) to the cuvette, mix well, incubate at 37°C for 5 min, and read the absorbance A1. Then add 250 μL of reagent R2, mix well, incubate at 37°C for 5 min, and read the absorbance A2. Finally, calculate ΔA (ΔA = A2 - A1), which is the measured absorbance value.

[0046] (4) Experimental Results and Data Analysis

[0047] 4.1 Test blank reagent

[0048] Table 2. Blank absorbance results

[0049] Time / min 0 1 2 2.5 3 3.5 4 4.5 5 ΔA / min absorbance value 0.018 0.018 0.019 0.018 0.019 0.02 0.02 0.02 0.02 0.0004

[0050] Test the blank absorbance. Test the blank sample at A505nm according to the operation in step (3). Repeat the measurement three times and the absorbance values ​​are 0.019, 0.018 and 0.020 respectively. It can be seen that the absorbance of the blank sample at A505nm is less than or equal to 0.05.

[0051] The blank absorbance was tested. At A505nm, the change in blank absorbance was tested within 5 minutes following the procedure in step (3). The test results are shown in Table 2, and the blank absorbance (ΔA / min) was 0.0004.

[0052] 4.2 Analytical Sensitivity

[0053] Table 3. Analysis of sensitivity test results

[0054]

[0055] Using the same batch of reagents, urine samples with a concentration of 50.3 U / L (meeting a concentration of (50±0.5) U / L) were tested according to the steps and methods described in steps (3) and (4) of Example 1. The test was repeated 3 times, and the absorbance values ​​changed as shown in Table 3, ranging from 0.20 to 0.30. It can be seen that 50 U / L of NAG can be accurately and repeatedly detected, indicating that the test method has high sensitivity.

[0056] 4.3 Test linear range

[0057] Urine samples with no obvious color and no NAG detected in the hospital were selected as the sample matrix. A series of standard concentrations of NAG samples were prepared according to the concentrations shown in Table 4, and the absorbance values ​​of the standard samples were tested according to step (3) of Example 1.

[0058] Table 4 Linearity Test Results

[0059]

[0060]

[0061] The test results are shown in Table 4. Then, a fitting curve was plotted with concentration on the x-axis and absorbance on the y-axis. The results are as follows: Figure 1 As shown, within the range of (0, 500 U / L), the linear correlation coefficient r between concentration and absorbance values ​​is ≥0.990. The linear range is wider than that of commercially available kits (0, 200 U / L). Furthermore, the linear deviation of the reagent provided by this invention does not exceed ±5 U / L within the range of (0, 50) U / L, and does not exceed ±10% within the range of [50, 500]. Simultaneously, based on the linear relationship between the measured absorbance and NAG (U / L) concentration, the NAG content (U / L) in the solution can be inferred by measuring the absorbance of the corresponding solution.

[0062] 4.4 Test repeatability

[0063] Table 5 Repeatability Test Results

[0064]

[0065] Using the same batch of reagents, urine samples with three different concentrations (low, medium, and high) were tested and calculated according to the steps and methods described in steps (3) and (4) of Example 1. The test groups were named Group 1, Group 2, and Group 3, and each group was tested 10 times. The test results are shown in Table 5: For urine samples or quality control materials with low, medium, and high concentrations, the coefficient of variation (CV) of the measured values ​​was ≤10% after 10 repeated measurements.

[0066] 4.5 Inter-batch variation in testing

[0067] Three batches of reagent kits were randomly selected and tested for low, medium and high concentration urine samples according to the steps and methods described in steps (3) and (4) of Example 1. The tests were performed in parallel for 3 times. The range of the test results with concentration values ​​in the range of (0, 50) U / L was calculated, and the relative range was calculated in the range of [50, 500].

[0068] Table 6. Results of inter-batch difference test

[0069]

[0070] The range of three randomly selected kits measured in the (0, 50) U / L range did not exceed ±5 U / L, and the relative range measured in the [50, 500] range did not exceed ±10%.

[0071] 4.6 Test spiked recovery rate

[0072] Table 7 Results of Spike Recovery Test

[0073]

[0074] Samples with three different concentrations (low, medium, and high) were taken, and a certain amount of standard was added. The concentration of the spiked samples was determined according to the steps and methods described in steps (3) and (4) of Example 1, and the spike recovery rate was calculated. If the sample spike recovery rate is between 90% and 110%, the recovery rate is considered good. The test results are shown in Table 7. The reagent provided by this invention has good spike recovery rates for samples with three different concentrations.

[0075] 4.7 Test anti-interference capability

[0076] Three different NAG concentrations (low, medium, and high) of urine samples were taken as the control group; then, the same samples were taken and the corresponding interfering factors were added to set up the corresponding interfering experimental groups to test the anti-interference ability of the reagents.

[0077] Table 8-1 Anti-interference capability

[0078]

[0079] Some of the interfering factors added are shown in Table 8-1, which are uric acid or bilirubin. Some of the interfering factors added are shown in Table 8-2, which are ascorbic acid, albumin, urea, lactic acid, GLU, triglycerides, or oxalic acid.

[0080] Then, using the reagents provided by this invention, the NAG content of the control group and each interference group named after the interference factor was tested and calculated according to the steps and methods described in steps (3) and (4) of Example 1.

[0081] The test results are shown in Tables 8-1 and 8-2. When ascorbic acid ≤340μmol / L, urea ≤42.9mmol / L, uric acid ≤1.4mmol / L, albumin ≤100mg / dL, bilirubin ≤342μmol / L, lactate ≤6.6mmol / L, glucose ≤55mmol / L, triglycerides ≤37mmol / L, and oxalic acid ≤81μmol / L, the standard deviation of this method in the (0, 50) U / L range does not exceed ±5U / L, and the relative standard deviation in the [50, 500] range does not exceed ±10%. It does not significantly interfere with the reagents. The test reagents and methods developed in this invention have strong anti-interference ability and good specificity.

[0082] Table 8-2 Anti-interference capability

[0083]

[0084] 4.8 Testing Accelerated Stability

[0085] Reagents R1 and R2 were subjected to accelerated aging in a 37°C oven. The corresponding reagents were taken at intervals as shown in Table 9, and the NAG content of samples with low, medium, and high concentrations was tested according to the steps and methods described in steps (3) and (4) of Example 1. The results of the aging test are shown in Table 9. Accelerated aging of reagents R1 and R2 at 37°C showed that, compared to the control, the standard deviation of the aged reagents in the (0, 50) U / L range did not exceed ±5 U / L, and the relative standard deviation in the [50, 500] U / L range did not exceed ±10%. The reagents showed good stability after 10 days of accelerated aging.

[0086] Table 9 Accelerated Aging Test at 37℃

[0087]

[0088]

[0089] Example 2: MNP-GlcNAc substrate method for determining NAG test values

[0090] Table 10 Linearity assessment data for the control kit

[0091]

[0092] A commonly available MNP-GlcNAc substrate assay kit was used as a control; its instructions show the following components:

[0093] Reagent R1: MNP-GlcNAc, HCl ≥ 0.1 mmol / L; citric acid ≥ 10 mmol / L.

[0094] Reagent R2: Sodium carbonate buffer ≥100mmol / L.

[0095] The testing procedure was performed according to the kit instructions. The linear range was tested, and the test data are shown in Table 10. The test data were analyzed, and the results are as follows: Figure 2 As shown: Figure 2 Curve 1 is the fitted curve within the linear range described in the specification; curve 2 is the fitted curve within the range (0, 300 U / L) expanded from the linear range described in the specification. When the linear range is expanded to (0, 300 U / L), the correlation of the curves decreases significantly. This kit cannot achieve the linear range (0, 500 U / L) measured in Example 1 using the kit provided by this invention. Figure 1 (As shown).

[0096] Example 3: VRA-NAG method for determining NAG content in the absence of ascorbic acid oxidase

[0097] (1) Reagent preparation.

[0098] Reagent R1:

[0099] Citric acid buffer solution: 100 mmol / L, pH 4.8; 5-[4-(3-methoxy-benzyl-raphanin)]-3-acetamide-N-acetamido-β-D-glucosidase (VRA-NAG) 15 mmol / L;

[0100] Surfactant: Triton X-100 0.1% (v / v);

[0101] Preservative: Potassium sorbate 0.1% (V / V);

[0102] Stabilizers: Magnesium chloride 10 mmol / L, EDTA 2 mmol / L, 1% (V / V) AES, 2.5% (V / V) HPD;

[0103] Protectant: Trehalose 5g / L.

[0104] Reagent R2:

[0105] Sodium carbonate buffer solution: 100 mmol / L, pH 10.2;

[0106] Surfactant: Triton X-100 0.1% (v / v);

[0107] Preservative: Proclin 300 0.1% (v / v);

[0108] (2) Measurement conditions. The measurement conditions are the same as those in step (2) of Example 1 of this patent.

[0109] (3) Operation steps. The operation steps are the same as those in steps (3) and (4) of Example 1 of this patent.

[0110] (3) Ascorbic acid interference investigation

[0111] The results of the ascorbic acid interference assay are shown in Table 11:

[0112] The reagent without added ascorbic acid oxidase has a weakened ability to resist ascorbic acid interference. When the ascorbic acid content in the sample is ≤170 μmol / L, there is no significant interference with the detection effect of the reagent; however, when the content is >170 μmol / L, significant interference occurs. In contrast, the detection formulation provided in Example 1 of this invention shows no significant interference with detection when the ascorbic acid content in the sample is ≤340 μmol / L. Therefore, the detection reagent provided in Example 1 of this invention has better anti-interference ability.

[0113] Table 11 Results of Anti-interference Ability Test

[0114]

[0115]

[0116] Example 4: VRA-NAG method for NAG content determination in the absence of stabilizer HPD and AES.

[0117] (1) Reagent preparation

[0118] Reagent R1:

[0119] Citric acid buffer solution: 100 mmol / L, pH 4.8;

[0120] VRA-NAG 15mmol / L;

[0121] Ascorbic acid oxidase 2 KU / L;

[0122] Surfactant: Triton X-100 0.1% (v / v);

[0123] Preservative: Potassium sorbate 0.1% (V / V);

[0124] Stabilizers: Magnesium chloride 10 mmol / L, EDTA 2 mmol / L;

[0125] Protectant: Trehalose 5g / L;

[0126] Reagent R2:

[0127] Sodium carbonate buffer solution: 100 mmol / L, pH 10.2;

[0128] Surfactant: Triton X-100 0.1% (v / v);

[0129] Preservative: Proclin 300 0.1% (V / V).

[0130] (2) Measurement conditions. The measurement conditions are the same as those in step (2) of Example 1 of this patent.

[0131] (3) Operation steps. The operation steps are the same as those in steps (3) and (4) of Example 1 of this patent.

[0132] (4) Accelerated stability testing. Reagents R1 and R2 were placed in a 37°C oven for accelerated aging. The NAG content of samples with low, medium, and high concentrations of the corresponding reagents was tested daily. Compared to the formulation provided in Example 1, the formulation in this example did not include the stabilizers AES and HPD. The aging test results in this example are shown in Table 12. Compared with Table 9 in Example 1, the accelerated stability of the formulation in this example is significantly lower than that of the formulation in Example 1 of this invention.

[0133] Table 12 Results of accelerated aging test at 37℃

[0134]

[0135] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An NAG detection kit, characterized in that, Including reagent R1 and reagent R2: Reagent R1 includes: Citric acid buffer solution: 100 mmol / L, pH 4.8; VRA-NAG: 15 mmol / L; Ascorbic acid oxidase: 2 KU / L; Surfactant: Triton X-100 0.1% (V / V); Preservative: Potassium sorbate 0.1% (V / V); Stabilizers: magnesium chloride 10 mmol / L, EDTA 2 mmol / L, 1% (V / V) AES and 2.5% (V / V) HPD; Protectant: Trehalose 5g / L; Reagent R2 includes: Sodium carbonate buffer solution: 100 mmol / L, pH 10.2; Surfactant: Triton X-100 0.1% (V / V); Preservative: Proclin 300 0.1% (V / V).

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