WO2022095576A1

WO2022095576A1 - NOVEL N-ACETYL-β-D GLUCOSAMINIDASE DETECTION AGENT

Info

Publication number: WO2022095576A1
Application number: PCT/CN2021/116609
Authority: WO
Inventors: 谢清华; 刘云海; 胡晓飞; 李久恩; 董雯
Original assignee: 山东博科生物产业有限公司
Priority date: 2020-11-09
Filing date: 2021-09-06
Publication date: 2022-05-12
Also published as: CN112501245B; CN112501245A

Abstract

An agent for detecting N-acetyl-β-D glucosaminidase. Said detection agent consists of R1 and R2. The agent R1 comprises a buffer solution, VRA-NAG, BSA, magnesium chloride, EDTA•2Na, Trixon-X100, SDS, sodium sulfite, polyethylene glycol 6000, ethylene glycol, glucose, beta cyclodextrin, 18-crown-6, and a preservative; and the agent R2 contains a buffer solution, Tween 20, urea, BSA, and a preservative. The agent uses magnesium ions as a substrate chelating agent for the first time, and the magnesium ions are chelated to VRA-NAG having a cyclic structure to form a six-membered cyclic chelate compound, thereby enhancing the stability of the agent.

Description

A Novel N-acetyl-β-D glucosaminidase detection reagent

technical field

The invention relates to a novel N-acetyl-β-D glucosaminidase detection reagent.

Background technique

N-acetyl-β-D-glucosaminidase (N-Acetyl-β-D-Glucosaminidase, NAG), also known as urease, is an acid hydrolase with a molecular weight of 140kD that exists in the lysosome of cells. The human body mainly exists in the proximal convoluted tubule epithelial cells of the kidney. Studies have shown that NAG is very sensitive to renal tubular injury, and more rapidly and directly reflects the degree of renal tubular active injury and changes in disease conditions than other low-molecular-weight proteins in urine, but it is very little in the urine of healthy people. In addition, using urine as a specimen is a non-invasive examination, which conforms to the trend of contemporary medical examination methods and is suitable for daily examination and continuous dynamic analysis. It can be seen that by measuring the activity of NAG in urine, it can be used for diabetes, various renal parenchymal diseases, drug and heavy metal nephrotoxicity, urinary tract infection (pyelonephritis), renal transplant rejection, hypertension and autoimmune diseases. The early detection of renal tubular injury has important clinical application value.

At present, NAG activity detection methods include radioimmunoassay, fluorescence analysis and visible spectrophotometry. Among them, the radioimmunoassay method and the fluorescence analysis method require expensive instruments, and have high requirements for the preparation of the substrate solution, which are not suitable for automated analysis of large clinical flow; and in the reported spectrophotometry methods, there is a problem of substrate dissolution. Problems such as poor performance or poor reagent stability make it difficult to meet clinical testing requirements.

SUMMARY OF THE INVENTION

In view of the deficiencies in the prior art, the present invention provides a detection reagent and a detection method with good stability and high sensitivity, which can be directly used without reconstitution.

A detection reagent for N-acetyl-β-D glucosaminidase, the detection reagent is composed of two reagents, reagent R1 and reagent R2,

Described reagent R1 comprises the component of following concentration:

The reagent R2 includes components in the following concentrations:

Preferably, the substrate is 5-[4-(3-methoxy-benzyl)]-raotanine-3-ammonium acetate-N-acetamido-β-D-glucoside (VRA-NAG).

Further, the BSA is bovine serum albumin, SDS is sodium dodecyl sulfate, and EDTA·2Na is disodium EDTA.

Preferably, the preservative is one of sodium azide, NaN ₃ and methylparaben.

Further, in the urine N-acetyl-β-D glucosaminidase detection reagent, the R1 buffer is a citric acid buffer with a pH of 15-30° C. and a pH of 4.0-6.0.

Further, in the urine N-acetyl-β-D glucosaminidase detection reagent, the buffer in the reagent R2 is a carbonate buffer with a pH of 15-30°C and a pH of 11.0-12.0.

Further, the described urine N-acetyl-β-D glucosaminidase detection reagent is used to detect the detection method of urine N-acetyl-β-D glucosaminidase, using an automatic biochemical analyzer to carry out the end point method. Measurement, the main detection wavelength is 505nm.

Further, the ratio of reagent R1 and reagent R2 is 3:1.

The basic principle of the present invention is as follows:

N-acetyl-β-D-glucosidase in urine converts 5-[4-(3-methoxy-benzyl)]-raodanin-3-ammonium acetate-N-acetyl Amino-β-D-glucoside (VRA-NAG) catalyzed hydrolysis to 5-[4-(3-methoxy-benzyl)]-raotannine-3-ammonium acetate (VRA), followed by the reaction of VRA in strong alkaline Allosteric produces fluorescence in the environment, the solution is purple-red, and there is an absorption peak at the wavelength of 505nm, and the activity of N-acetyl-β-D glucosidase can be determined by detecting the change of the absorbance at the wavelength of 505nm.

Note: VRA-NAG: 5-[4-(3-methoxy-benzyl)]-raotanning-3-ammonium acetate-N-acetylamino-β-D-glucoside, molecular weight 545.

NAG: N-acetyl-β-D glucosidase

VRA: 5-[4-(3-Methoxy-benzyl)]-Raotannin-3-ammonium acetate

Beneficial effects of the present invention:

1) Using the new 5-[4-(3-methoxy-benzyl)]-raotanning-3-ammonium acetate-N-acetylamino-β-D-glucoside (VRA-NAG) as the reaction substrate , which not only solves the problem of large interference of reagent chromogens, but also significantly enhances the solubility of substrates and the accuracy of reagents;

2) The present invention uses magnesium ion as a substrate chelating agent for the first time, and magnesium ion chelates with VRA-NAG having a cyclic structure and forms a six-atom ring chelate, which enhances the stability of the reagent;

3) The reagent has good accuracy and stability, strong anti-interference, easy to use, and meets the needs of clinical batch testing.

Description of drawings

Fig. 1 is the correlation curve diagram of Example 1 in the detection reagent of N-acetyl-β-D glucosaminidase of the present invention;

Fig. 2 is the correlation graph of Example 2 in the detection reagent of N-acetyl-β-D glucosaminidase of the present invention;

Fig. 3 is the correlation graph of Example 3 in the detection reagent of N-acetyl-β-D glucosaminidase of the present invention;

4 is a graph showing the validity period stability of Examples 1-3 and a comparative example in the detection reagent for N-acetyl-β-D glucosaminidase of the present invention.

Detailed ways

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will describe in detail with reference to specific embodiments and accompanying drawings.

Example 1:

The detection reagent for N-acetyl-β-D glucosidase in this embodiment includes reagent R1 and reagent R2, wherein:

The composition and content of reagent R1 are as follows:

The composition and content of reagent R2 are as follows:

How to use this test reagent:

The urine N-acetyl-β-D glucosidase detection reagent described in this example should be detected by an automatic biochemical analyzer with dual-reagent function, such as Hitachi 7180 automatic analyzer, Hitachi 7080 automatic analyzer and Mindray 800, etc., use the endpoint method for determination, and set the detection main wavelength to 505nm. Place the detection reagents R1 and R2 on the corresponding reagent positions in a ratio of 3:1, and place double distilled water, calibrators, quality control products and samples to be tested on the sample tray. The operations are shown in Table 1:

Table 1 Example 1 Reagent Detection Method

Calculation formula: urine N-acetyl-β-D glucosidase concentration (U/L)=(sample ΔA÷calibrator ΔA)×calibrator concentration.

Example 2:

The detection reagent of N-acetyl-β-D-glucosidase of the present embodiment comprises reagent R1 and reagent R2, wherein:

The composition and content of reagent R1 are as follows:

The composition and content of reagent R2 are as follows:

The use method and detection method are the same as those in Example 1.

Example 3:

The composition and content of reagent R1 are as follows:

The composition and content of reagent R2 are as follows:

The use method and detection method are the same as those in Example 1.

Example 4:

Interference experiment: take fresh mixed urine, divide it into 2 equal parts, then divide each equal part into 6 equal parts, add different interfering substances, so that the concentration in the urine reaches the requirements of Table 2. Then use the reagents obtained in the examples respectively, and the urine N-acetyl-β-D-glucosidase detection reagents that are common and recognized in the market as the control group to measure the NAG content in the urine at the same time. The measurement results of each group after substances are shown in Table 2.

Relative deviation (%)=(measured mean value of interference sample−measured mean value of control sample)/measured mean value of control sample×100%.

Table 2 Comparison of anti-interference performance of example reagents

As can be seen from Table 2, the detection reagents of Examples 1-3 are when glucose≤20mmol/L, bilirubin≤40mg/dL, hemoglobin≤200mg/dL, triglyceride≤500mg/dL and ascorbic acid≤50mg/dL There was no significant interference with the test results. In contrast, the reagents of the control group were significantly interfered in the presence of the above-mentioned concentration of interfering substances, which indicated that by using the new substrate 5-[4-(3-methoxy-benzyl)]-raotanning-3-ammonium acetate-N- Acetylamino-β-D-glucoside (VRA-NAG) is used as the reaction substrate, and magnesium ion is used as a substrate chelator, which can effectively strengthen the anti-interference ability of the detection reagent and avoid interference such as glucose, bilirubin and ascorbic acid. , the anti-interference performance of the reagent in the example group was significantly improved, far superior to the control reagent.

Example 5:

Correlation experiment: use the reagent formula described in the embodiment to prepare the detection reagent, and carry out comparative detection with the N-acetyl-β-D glucosidase detection kit of a company recognized by the State Food and Drug Administration, which is common in the market. The groups tested 40 clinical urine samples respectively, and the test results are shown in Table 3. And obtained the correlation curve of each example and the control group (as shown in Figure 1), the test results showed that the correlation coefficient between Example 1 and the control group was 0.9979, and the correlation coefficient between Example 2 and the control group was 0.9949. The correlation coefficient between Example 3 and the control group was 0.9962, and the relative deviations of 40 samples of the reagents in each example group and the control group were all ≤10%, indicating that Examples 1-3 groups had great correlation with the control group.

Table 3 embodiment reagents and the market common and approved urine N-acetyl-β-D glucosidase detection kit comparative detection results

Example 6:

Reagent stability comparison test: For the reagents in Examples 1-3, each group of examples was evenly divided into 13 groups, and the filling volume of each group: Reagent R1 was 18mL, and Reagent R2 was 6mL; and 13 groups were commonly used in the market. The N-acetyl-β-D glucosidase detection kit of a company approved by the State Food and Drug Administration was used as a control. Put it in a refrigerator at 2-8°C, and take out a set of reagents on the same day of every month to detect NAG quality control products (target value is 25.8 μmol/L). The test results are shown in Figure 2. It is more stable than the common N-acetyl-β-D glucosidase detection kit in the market under the storage condition of 8 ℃.

Through verification, this reagent has good correlation with similar detection reagents, and the results of clinical test samples are consistent, which can meet the application requirements of the market for products, and has good anti-interference performance. It is a more stable and good N-acetyl-β-D Glucosidase detection reagent.

Claims

A urine N-acetyl-β-D glucosaminidase detection reagent, the detection reagent is composed of two reagents, a reagent R1 and a reagent R2, and is characterized in that:

The reagent R1 contains the following concentrations of components:

The reagent R2 contains the following concentrations of components:
The urine N-acetyl-β-D glucosaminidase detection reagent according to claim 1, wherein the citric acid buffer in the reagent R1 is 15-30°C, and the pH is 4.0-6.0.
The urine N-acetyl-β-D glucosaminidase detection reagent according to claim 1, wherein the carbonate buffer in the reagent R2 is 15-30°C, and the pH is 11.0-12.0.
The urine N-acetyl-β-D glucosaminidase detection reagent according to claim 1, wherein the preservative is one of sodium azide, NaN 3 , methylparaben or several.