CN111826417B - N-acetyl-beta-D-glucosaminidase detection kit with good stability, preparation method and application - Google Patents
N-acetyl-beta-D-glucosaminidase detection kit with good stability, preparation method and application Download PDFInfo
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Abstract
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.
Description
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 application thereof.
Background
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 under normal conditions, NAG in serum cannot be excreted from urine by glomerular filtration. 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:
30-120mmol/L of citric acid, 50-150mmol/L of sodium acetate, 0.5-20mmol/L of ionic preparation, 1-10KU/L, PC-300:1-5ml/L of ascorbic acid oxidase and pH =4.80-5.20.
Further, in the reagent 2, the concentrations of the components are as follows:
and TIRS:50-200mmol/L, stabilizer: 10-100g/L, potassium ferrocyanide: 0.05-1.5g/L, surfactant: 0.5-5g/L, MPT-NAG:45-150mmol/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:
30mmol/L citric acid, 50mmol/L sodium acetate, 1.0mmol/L ionic agent, 5KU/L, PC-300:1ml/L ascorbic acid oxidase, pH =4.80;
in the reagent 2, the concentrations of the components are as follows:
and TIRS:50mmol/L, stabilizer: 10g/L, potassium ferrocyanide: 0.1g/L, surfactant: 1g/L, MPT-NAG:50mmol/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 comprises any one of glycerol, 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:
30-120mmol/L of citric acid, 50-150mmol/L of sodium acetate, 0.5-20mmol/L of ionic preparation and 1-10KU/L, PC-300:1-5ml/L of ascorbic acid oxidase;
preparing raw materials TIRS, a stabilizer, potassium ferrocyanide, a surfactant, MPT-NAG, PC-300 and lipoprotein esterase of the reagent 2 into a solution, and adjusting the pH value to 7.4-7.9 by adopting acid liquid and active alkali liquid to obtain a reagent 2 with the concentration of each component:
and TIRS:50-200mmol/L, stabilizer: 10-100g/L, potassium ferrocyanide: 0.05-1.5g/L, surfactant: 0.5-5g/L, MPT-NAG:45-150mmol/L, PC-300:1-5ml/L, lipoprotein esterase: 1-10KU/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 at least have the following technical effects or advantages:
the kit disclosed by the invention can effectively resist interference by adding ascorbic acid oxidase, potassium ferrocyanide and 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, better in anti-interference capability and higher in sensitivity.
Detailed Description
The present invention will be specifically explained below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented thereby. 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:
30-120mmol/L of citric acid, 50-150mmol/L of sodium acetate, 0.5-20mmol/L of ionic preparation, 1-10KU/L, PC-300:1-5ml/L of ascorbic acid oxidase and pH =4.80-5.20.
Further, in the reagent 2, the concentrations of the components are as follows:
and (3) TIRS:50-200mmol/L, stabilizer: 10-100g/L, potassium ferrocyanide: 0.05-1.5g/L, surfactant: 0.5-5g/L, MPT-NAG:45-150mmol/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.
MPT-NAG+H2O
N-acetyl-beta-D-glucosamine + MPT
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 PEG 20000) 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.
Examples
The present invention was provided with 6 comparative examples and 1 example.
Comparative example 1: in a traditional kit using 5- [4- (3-methoxy-benzyl-rhodanine) ] -3-ammonium acetate-N-acetamido-beta-D-glucoside as a substrate, a reagent is prepared by referring to a comparison document CN 105203533B;
comparative example 2: the substrate MPT-NAG is placed in R1.
Comparative example 3: a lack of ethylene glycol;
comparative example 4: absence of Tritox-405;
comparative example 5 pH of R2 was 8.5;
comparative example 6: the pH of R2 was 6.5.
1. The components of the comparative and example kits are shown in table 1:
TABLE 1 ingredient Table for comparative examples and example kits
2. Performance evaluation: the following tests were carried out for the above comparative examples and examples, respectively:
(1) Precision: repeatedly measuring the same sample for 20 times, and calculating CV (%), wherein the CV is less than or equal to 3.4% to meet the requirement;
(2) Linear range: measuring a range sample of 5-1000U/L, calculating a correlation coefficient, wherein r is less than or equal to 0.9900 to meet the requirement;
(3) Accuracy: measuring the third-party quality control product, and calculating the deviation between the measured average value and the target value, wherein the deviation is less than or equal to 10 percent and meets the requirement;
(4) Functional sensitivity: and (3) determining low-concentration samples with NAG concentration of 1U/L, 2.5U/L, 5U/L and 10U/L, determining each sample 10 times, calculating a mean value, a standard deviation, a deviation and a coefficient of variation, wherein CV is less than 10% and the deviation is less than 10% so as to meet the use requirement.
(5) Stability: the reagent was left at 37 ℃ for 7 days, and the above indices were evaluated: precision, linear range, accuracy, functional sensitivity;
(6) Interference evaluation:
diluting the interference substances with purified water, adding the diluted interference substances into blank control serum according to the proportion of 1:9 (the blank control serum is mixed serum of normal people, no interference substances are added, and the value is near the medical decision level) so that the final concentration after adding the serum meets the concentration to be evaluated, then respectively measuring the blank serum and the serum added with the interference substances with different concentrations by using a comparative example group and an example group, measuring each sample for 3 times, calculating the average value, and calculating the deviation from the measured value of the blank control serum, namely the interference degree. And if the interference degree is less than 10%, judging the anti-interference state, otherwise, judging the non-anti-interference state.
The general interferents and concentrations in serum were: 1.0g/L ascorbic acid, 0.5g/L bilirubin and less than or equal to 10g/L triglyceride.
The test results are shown in tables 2 and 3:
TABLE 2 interference free data
Table 3 summary of performance evaluation data
Table 4 summary of sensitivity data
After 7 days of heat breaking of the reagent:
summary of sensitivity data before and after thermal disruption in examples 1 and 2 of the present invention
From the above experimental results, it can be seen that the anti-interference ability of the reagents of each comparative example and example was almost the same before the thermal disruption, but after the thermal disruption, the anti-interference ability was not affected compared with that of comparative example 4 (after the thermal disruption of comparative example 4) and examples 1 and 2. The accuracy of the reagent after thermal disruption was poor due to poor stability before and after thermal disruption as compared with control 3, but the accuracy was poor as compared with control 4, and the analytical sensitivity was not as good as in examples 1 and 2 before and after thermal disruption. The anti-interference capability of the comparison group 5 and the comparison group 6 is reduced after thermal damage, and the linearity and the sensitivity are influenced after thermal damage.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While the preferred embodiments of the invention have been described, those skilled in the art will appreciate that once the basic inventive concepts have been developed, additional variations and modifications may be made to the embodiments. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (4)
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;
in the reagent 1, the concentrations of the components are as follows:
citric acid: 30-120mmol/L, sodium acetate: 50-150mmol/L, ionic preparation: 0.5-20mmol/L, ascorbic acid oxidase: 1-10KU/L, PC-300:1-5ml/L, pH =4.80-5.20;
in the reagent 2, the concentrations of the components are as follows:
and (3) TIRS:50-200mmol/L, stabilizer: 10-100g/L, potassium ferrocyanide: 0.05-1.5g/L, surfactant: 0.5-5g/L, MPT-NAG:45-150mmol/L, PC-300:1-5ml/L, lipoprotein esterase: 1-10KU/L, pH =7.4-7.9;
the ionic preparation 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.
2. 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:
and (3) citric acid: 30mmol/L, sodium acetate: 50mmol/L, ionic preparation: 1.0mmol/L, 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:50mmol/L, stabilizer: 10g/L, potassium ferrocyanide: 0.1g/L, surfactant: 1g/L, MPT-NAG:50mmol/L, PC-300:1ml/L, lipoprotein esterase: 3KU/L, pH =7.4.
3. The method for preparing the N-acetyl-beta-D-glucosaminidase detection kit with good stability as defined in any one of claims 1-2, comprising:
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 using an acid liquid active alkali solution to obtain the reagent 1 with the component concentration as claimed in claim 1 or 2;
preparing a solution from the raw materials of the reagent 2, namely TIRS, a stabilizer, potassium ferrocyanide, a surfactant, MPT-NAG, PC-300 and lipoprotein esterase, 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 1 or 2.
4. The method for preparing the N-acetyl-beta-D-glucosaminidase detection kit of claim 3, wherein the acid solution is hydrochloric acid, and the alkali solution is NaOH solution.
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