阅读说明：本技术 一种稳定性好的N-乙酰-β-D-氨基葡萄糖苷酶检测试剂盒、制备方法及用途 (N-acetyl-beta-D-glucosaminidase detection kit with good stability, preparation method and application ) 是由 龚婷 吴年芬 梁艳 张超 张雪娇 舒芹 赵畅 于 2020-08-04 设计创作，主要内容包括：本发明提供了一种稳定性好的N-乙酰-β-D-氨基葡萄糖苷酶检测试剂盒,属于生物检测技术领域,包括试剂1和试剂2,所述试剂1包括柠檬酸、乙酸钠、离子制剂、抗坏血酸氧化酶和PC-300；所述试剂2包括TIRS、稳定剂、亚铁氰化钾、表面活性剂、MPT-NAG、PC-300和脂蛋白酯酶。以6-甲基-2-硫代吡啶-N-乙酰-β-D-葡萄糖苷为底物,选择合适的反应体系、缓冲液和pH值,可以让底物更加稳定、抗干扰能力更好、灵敏度更高。本发明还提供了一种稳定性好的N-乙酰-β-D-氨基葡萄糖苷酶检测试剂盒的制备方法及用途。(The invention provides an N-acetyl-beta-D-glucosaminidase detection kit with good stability, which belongs to the technical field of biological detection and comprises a reagent 1 and a reagent 2, wherein the reagent 1 comprises citric acid, sodium acetate, an ionic preparation, ascorbic acid oxidase and PC-300; the reagent 2 comprises TIRS, a stabilizer, potassium ferrocyanide, a surfactant, MPT-NAG, PC-300 and lipoprotein esterase. The 6-methyl-2-thiopyridine-N-acetyl-beta-D-glucoside is used as a substrate, and a proper reaction system, a buffer solution and a pH value are selected, so that the substrate is more stable, the anti-interference capability is better, and the sensitivity is higher. The invention also provides a preparation method and application of the N-acetyl-beta-D-glucosaminidase detection kit with good stability.)

1. The N-acetyl-beta-D-glucosaminidase detection kit with good stability is characterized by comprising a reagent 1 and a reagent 2, wherein the reagent 1 comprises citric acid, sodium acetate, an ionic preparation, ascorbic acid oxidase and PC-300; the reagent 2 comprises TIRS, a stabilizer, potassium ferrocyanide, a surfactant, MPT-NAG, PC-300 and lipoprotein esterase.

2. The N-acetyl-beta-D-glucosaminidase detection kit that has good stability according to claim 1, characterized in that, in the reagent 1, the concentration of each component is:

citric acid: 30-120moml/L, sodium acetate: 50-150 moll/L, ionic preparation: 0.5-20moml, ascorbic acid oxidase: 1-10KU/L, PC-300: 1-5ml/L, pH 4.80-5.20.

3. The kit for detecting N-acetyl-beta-D-glucosaminidase with good stability according to claim 1, characterized in that, in the reagent 2, the concentrations of the components are as follows:

and TIRS: 50-200 mol/L, stabilizer: 10-100g/L, potassium ferrocyanide: 0.05-1.5g/L, surfactant: 0.5-5g/L, MPT-NAG: 45-150moml/L, PC-300: 1-5ml/L, lipoprotein esterase: 1-10KU/L, pH 7.4-7.9.

4. The kit for detecting N-acetyl-beta-D-glucosaminidase with good stability of claim 1, characterized in that,

in the reagent 1, the concentrations of the components are as follows:

citric acid: 30moml/L, sodium acetate: 50 moll/L, ionic preparation: 1.0moml, ascorbic acid oxidase: 5KU/L, PC-300: 1ml/L, pH 4.80;

in the reagent 2, the concentrations of the components are as follows:

and TIRS: 50 moll/L, stabilizer: 10g/L, potassium ferrocyanide: 0.1g/L, surfactant: 1g/L, MPT-NAG: 50moml/L, PC-300: 1ml/L, lipoprotein esterase: 3KU/L, pH 7.4.

5. The N-acetyl-beta-D-glucosaminidase detection kit of claim 1 wherein the ionic agent comprises any one or more of sodium chloride, potassium sulfate, sodium sulfate, magnesium chloride and magnesium sulfate.

6. The kit for detecting N-acetyl-beta-D-glucosaminidase with good stability as claimed in claim 1, wherein the surfactant comprises any one of Tritox-405, Emulgen A60, Triton-100 and GENAPOLX-080.

7. The kit for detecting N-acetyl-beta-D-glucosaminidase with good stability as claimed in claim 1, wherein the stabilizer comprises any one of glycerol, ethylene glycol, PEG6000, PEG8000 and PEG 20000.

8. The method for preparing the N-acetyl-beta-D-glucosaminidase detection kit with good stability as defined in any one of claims 1-7, comprising:

preparing raw materials of citric acid, sodium acetate, an ionic preparation, ascorbic acid oxidase and PC-300 of the reagent 1 into a solution, and adjusting the pH value to 4.80-5.20 by using an acid liquid and an active alkali liquid to obtain the reagent 1 with the component concentration as claimed in claim 2;

preparing a solution from the raw materials TIRS, a stabilizer, potassium ferrocyanide, a surfactant, MPT-NAG, PC-300 and lipoprotein esterase of the reagent 2, and adjusting the pH value to 7.4-7.9 by using an acid liquid and an active alkali liquid to obtain the reagent 2 with the component concentration as defined in claim 3.

9. The method for preparing the N-acetyl-beta-D-glucosaminidase detection kit of claim 8, wherein the acid solution is hydrochloric acid and the alkali solution is NaOH solution.

10. Use of the kit of any one of claims 8 in an N-acetyl- β -D-glucosaminidase assay.

Technical Field

The invention belongs to the technical field of biological detection, and relates to an N-acetyl-beta-D-glucosaminidase detection kit with good stability, a preparation method and an application background technology

N-acetyl-beta-D-glucosaminidase (NAG) is an acid hydrolase, also called urease, located in lysosomes and has a relative molecular weight of about 140000. It is widely distributed in human tissues, but is most abundant in prostate and renal proximal tubules. NAG has a molecular weight of about 130-140 KD, and NAG in serum cannot be excreted from urine by glomerular filtration under normal conditions. Elevation of NAG in urine is an early manifestation of kidney disease and is a sensitive indicator of tubular damage. In renal transplant patients, rejection can be detected early by urinary NAG measurement, and the NAG in the urine is increased 1-3 days before clinical indication. There is an increasing research on the use of late 80 s in the area of diabetic nephropathy, where it is noteworthy that the increase in urinary NAG may precede the increase in mALb in the early stages of diabetic nephropathy, suggesting that diabetic tubular damage may precede glomerular damage. A new idea has emerged to claim mALb and tubular marker proteins (NAG etc.) as routine indicators of early detection and monitoring of diabetic complications. In addition, the monitoring of NAG in urine has different degrees of elevation in various renal parenchymal diseases, is a sensitive indicator of kidney damage, and the elevation is seen in acute and chronic nephritis, renal failure caused by shock, nephrotic syndrome, toxic nephropathy and the like. Research shows that when the renal tubular injury is caused by diabetes, hypertensive glomerular early injury, drug nephrotoxicity, infection shock, renal transplantation rejection reaction and the like, the activity of NAG in urine is obviously increased and is earlier than other urinary enzymes, so the method has great value for early diagnosis of the renal tubular injury.

At present, NAG determination is the urine enzymology diagnostic item with the highest application rate, the reaction of the NAG determination to renal tubular injury is sensitive, and in addition, urine is used as a specimen, the NAG determination is a non-invasive inspection, is suitable for daily inspection and continuous dynamic analysis, and is also convenient for people to screen. The determination method of NAG in clinical application mainly comprises an radioimmunoassay, a fluorescence analysis method, an ultraviolet-visible spectrophotometry and the like, wherein the spectrophotometry takes the synthesis of a chromogen substrate as a main methodology. The substrate is decomposed by NAG, and the concentration of NAG is determined by measuring the absorbance of the decomposition product of the substrate at a specific wavelength. In the market, 5- [4- (3-methoxy-phenmethyl-rhodanine) ] -3-ammonium acetate-N-acetamido-beta-D-glucoside is generally adopted as a substrate, but a reagent prepared by using the substrate generally has the defects of poor stability, low analysis sensitivity and the like.

Disclosure of Invention

In order to solve the technical problems of poor stability and low sensitivity of the existing NAG detection reagent, the invention provides an N-acetyl-beta-D-glucosaminidase detection kit with good stability, which selects 6-methyl-2-thiopyridine-N-acetyl-beta-D-glucoside as a substrate and is matched with a proper reaction system, a buffer solution and a pH value, so that the substrate is more stable, the anti-interference capability is better and the sensitivity is higher.

The invention also provides a preparation method and application of the N-acetyl-beta-D-glucosaminidase detection kit with good stability.

The invention is realized by the following technical scheme:

an N-acetyl-beta-D-glucosaminidase detection kit with good stability comprises a reagent 1 and a reagent 2, wherein the reagent 1 comprises citric acid, sodium acetate, an ionic preparation, ascorbic acid oxidase and PC-300; the reagent 2 comprises TIRS, a stabilizer, potassium ferrocyanide, a surfactant, MPT-NAG, PC-300 and lipoprotein esterase.

Wherein, in the reagent 1, the concentrations of the components are as follows:

citric acid: 30-120moml/L, sodium acetate: 50-150 moll/L, ionic preparation: 0.5-20moml, ascorbic acid oxidase: 1-10KU/L, PC-300: 1-5ml/L, pH 4.80-5.20.

Further, in the reagent 2, the concentrations of the components are as follows:

and TIRS: 50-200 mol/L, stabilizer: 10-100g/L, potassium ferrocyanide: 0.05-1.5g/L, surfactant: 0.5-5g/L, MPT-NAG: 45-150moml/L, PC-300: 1-5ml/L, lipoprotein esterase: 1-10KU/L, pH 7.4-7.9.

Preferably, in the reagent 1, the concentrations of the components are as follows:

citric acid: 30moml/L, sodium acetate: 50 moll/L, ionic preparation: 1.0moml, ascorbic acid oxidase: 5KU/L, PC-300: 1ml/L, pH 4.80;

in the reagent 2, the concentrations of the components are as follows:

and TIRS: 50 moll/L, stabilizer: 10g/L, potassium ferrocyanide: 0.1g/L, surfactant: 1g/L, MPT-NAG: 50moml/L, PC-300: 1ml/L, lipoprotein esterase: 3KU/L, pH 7.4.

Further, the ionic agent comprises any one or more of sodium chloride, potassium sulfate, sodium sulfate, magnesium chloride and magnesium sulfate.

Further, the surfactant includes any one of Tritox-405, Emulgen A60, Triton-100 and GENAPOLX-080.

Further, the stabilizer includes any one of glycerin, ethylene glycol, PEG6000, PEG8000 and PEG 20000.

A preparation method of an N-acetyl-beta-D-glucosaminidase detection kit with good stability comprises the following steps:

preparing raw materials of citric acid, sodium acetate, an ionic preparation, ascorbic acid oxidase and PC-300 of a reagent 1 into a solution, and adjusting the pH value to 4.80-5.20 by adopting an acid liquid active alkali solution to obtain the reagent 1, wherein the concentration of each component of the obtained reagent 1 is as follows:

citric acid: 30-120moml/L, sodium acetate: 50-150 moll/L, ionic preparation: 0.5-20moml, ascorbic acid oxidase: 1-10KU/L, PC-300: 1-5 ml/L;

preparing a solution from the raw materials TIRS, a stabilizer, potassium ferrocyanide, a surfactant, MPT-NAG, PC-300 and lipoprotein esterase of the reagent 2, and adjusting the pH value to 7.4-7.9 by adopting an acid liquid and an active alkali liquid to obtain a reagent 2 with the concentration of each component:

and TIRS: 50-200 mol/L, stabilizer: 10-100g/L, potassium ferrocyanide: 0.05-1.5g/L, surfactant: 0.5-5g/L, MPT-NAG: 45-150moml/L, PC-300: 1-5ml/L, lipoprotein esterase: 1-10 KU/L.

Further, the acid solution is hydrochloric acid, and the alkali solution is a NaOH solution.

An application of an N-acetyl-beta-D-glucosaminidase detection kit with good stability in N-acetyl-beta-D-glucosaminidase detection.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

the kit disclosed by the invention has the advantages that the anti-interference effect can be effectively realized by adding the ascorbic acid oxidase, the potassium ferrocyanide and the lipoprotein esterase, the stability of the reagent is enhanced by adding the stabilizer, the shelf life of the reagent is prolonged, the reaction of the reagent can be accelerated by adding the ionic agent and the surfactant, and the obtained kit can enable a substrate to be more stable, has better anti-interference capability and higher sensitivity.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

In order to solve the technical problems, the embodiment of the invention provides the following general ideas:

the invention adopts a 6-methyl-2-thiopyridine-N-acetyl-beta-D-glucosaccharase (MPT) substrate method to detect the activity of N-acetyl-beta-D-glucosaccharase in human serum or urine, and the reagent is stable for a long time, high in sensitivity and convenient to detect. However, the anti-interference capability of the existing MPT-NAG detection reagent is reduced after thermal damage, and the invention aims to solve the problem that the reagent can still keep good anti-interference capability after thermal damage.

Based on this, the substrate MPT-NAG is put into the reagent 2, and a proper reaction system, buffer solution and pH value are selected, so that the substrate is more stable, the anti-interference capability is better, and the sensitivity is higher.

Specifically, the N-acetyl-beta-D-glucosaminidase detection kit with good stability comprises a reagent 1 and a reagent 2, wherein the reagent 1 comprises citric acid, sodium acetate, an ionic preparation, ascorbic acid oxidase and PC-300; the reagent 2 comprises TIRS, a stabilizer, potassium ferrocyanide, a surfactant, MPT-NAG, PC-300 and lipoprotein esterase.

Wherein, in the reagent 1, the concentrations of the components are as follows:

citric acid: 30-120moml/L, sodium acetate: 50-150 moll/L, ionic preparation: 0.5-20moml, ascorbic acid oxidase: 1-10KU/L, PC-300: 1-5ml/L, pH 4.80-5.20.

Further, in the reagent 2, the concentrations of the components are as follows:

and TIRS: 50-200 mol/L, stabilizer: 10-100g/L, potassium ferrocyanide: 0.05-1.5g/L, surfactant: 0.5-5g/L, MPT-NAG: 45-150moml/L, PC-300: 1-5ml/L, lipoprotein esterase: 1-10KU/L, pH 7.4-7.9.

The ionic agent comprises any one or more of sodium chloride, potassium sulfate, sodium sulfate, magnesium chloride and magnesium sulfate. The surfactant comprises any one of Tritox-405, Emulgen A60, Triton-100 and GENAPOLX-080. The stabilizer comprises any one of glycerol, glycol, PEG6000, PEG8000 and PEG 20000.

The invention adopts an MPT-NAG detection method, N-acetyl-beta-D-glucosaminidase (NAG) in a sample catalyzes a substrate 6-methyl-2-thiopyridine-N-acetyl-beta-D-glucoside (MPT-NAG) to hydrolyze to generate 6-methyl-2-thiopyridine (MPT), and the absorbance rising rate of the MPT at a specific wavelength is in positive correlation with the NAG activity.

In the invention, the ascorbic acid oxidase must be added into the reagent 1, and when the reagent 1 is incubated with serum, the interference of vitamin C in the serum can be effectively cleared before the reagent 2 is added.

Potassium ferrocyanide must be added into the reagent 2, the potassium ferrocyanide cannot coexist with ASO enzyme, and the interference of bilirubin in serum on the measured value of the reagent can be effectively eliminated when the potassium ferrocyanide is added into the reagent 2.

The ionic preparation can effectively activate enzyme and substrate in the reagent and accelerate the reaction of the reagent.

Surfactant (b): tritox-405, Emulgen A60, Triton-100 and GENAPOLX-080 can act together with a stabilizer to completely dissolve substrates and accelerate reagent reaction.

The stabilizer (glycerol, glycol, PEG6000, PEG8000 and PEG20000) can act together with the surfactant to completely dissolve the substrate, accelerate the reaction of the reagent, stabilize the substrate in the reagent and prolong the shelf life of the reagent. The substrate 6-methyl-2-thiopyridine-N-acetyl-beta-D-glucoside needs to be placed in the reagent 2 to ensure that the reagent is more stable.

The interference of triglyceride can be eliminated by adding lipoprotein esterase into the reagent.

The kit can effectively resist interference by adding ascorbic acid oxidase, potassium ferrocyanide and lipoprotein esterase, enhances the stability of the reagent by adding a stabilizer, prolongs the quality guarantee period of the reagent, can accelerate the reaction of the reagent by adding an ionic preparation and a surfactant, and can ensure that a substrate is more stable, the anti-interference capability is better and the sensitivity is higher.

The following will explain in detail an N-acetyl- β -D-glucosaminidase detection kit with good stability in the present application with reference to examples, comparative examples and experimental data.