CN113252587A - NAG detection kit and application thereof - Google Patents
NAG detection kit and application thereof Download PDFInfo
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Abstract
The invention discloses an NAG detection kit and application thereof. Specifically discloses a kit for detecting N-acetyl-beta-D-glucosaminidase (NAG), which comprises a reagent R1 and a reagent R2; the reagent R1 comprises: 10-500 mmol/L buffer solution A, pH 4-6; 0.1-5 mmol/L of substrate; 1-5 g/L chloride; 1-5 g/L of EDTA or sodium salt thereof; 0.1-5 g/L stabilizer; 0.1-5 mmol/L of substrate approximation compound; 0.1-5% of surfactant, wherein the% is the volume percentage of the reagent R1; 0.1-5 g/L of preservative; the substrate is one or more of VRA-NAG, PNP-NAG and CNP-NAG. The NAG detection kit has high bottle opening stability and transportation stability.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to an NAG detection kit and application thereof.
Background
N-acetamido-beta-D-glucosidase (NAG) is a comparatively important lysosomal hydrolase in the human body, which is widely present in the human body, and the damage to renal function caused by various reasons can increase the NAG index value, which can very sensitively reflect the damage of the kidney. In recent years, urease typified by NAG is ideal for detecting kidney diseases, and the measurement method is simple and easy to implement, and has clinical practicability. The relative molecular mass of NAG is 130-140 KD, the half-life period in plasma is only 5 minutes, and NAG in plasma can not be filtered by glomerulus. Elevated urinary NAG, which is a sensitive and specific indicator reflecting tubular damage, predicts damage to proximal tubular epithelial cells and release of NAG by lysosomal rupture.
At present, a plurality of methods for detecting NAG in urine mainly comprise 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. The reagent prepared by the existing substrate on the market generally has the defects of unstable bottle opening, unstable transportation and the like.
Disclosure of Invention
In order to solve the defects of unstable bottle opening, unstable transportation and the like of a kit for NAG detection in the prior art, the invention provides the NAG detection kit and the application thereof.
The invention provides a NAG detection kit in a first aspect, which comprises a reagent R1 and a reagent R2; the reagent R1 comprises: 10-500 mmol/L buffer solution A, pH 4-6; 0.1-5 mmol/L of substrate; 1-5 g/L chloride; 1-5 g/L of EDTA or sodium salt thereof; 0.1-5 g/L stabilizer; 0.1-5 mmol/L of substrate approximation compound; 0.1-5% of surfactant, wherein the% is the volume percentage of the reagent R1; 0.1-5 g/L of preservative; the substrate is one or more of VRA-NAG, PNP-NAG and CNP-NAG; the reagent R2 comprises: 10-500 mmol/L buffer solution B with the pH value of 10-12; 0.1-5 g/L preservative.
In the present invention, the term "substrate proximity" may refer to a compound that has a structure close to that of a substrate hydrolysate.
The NAG is N-acetyl-beta-D-glucosaminidase.
The VRA-NAG is 5- [4- (3-methoxy-benzyl-rhodanine) ] -3-ammonium acetate-N-acetamido-beta-D-glucoside, and the cas number is 125261-87-2; the PNP-NAG is p-nitrobenzene-N-acetamido-beta-D-glucosaminide, and the cas number is 3459-18-5; the CNP-NAG is 2-chloro-4-nitrophenyl-N-acetyl-beta-D-glucosaminide with the cas number of 103614-82-0.
The substrate mimetic is preferably one or more of N-acetyl-D-mannosamine, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine.
Preferably, the buffer solution A is one or more of citric acid buffer solution, potassium hydrogen phthalate, citric acid-disodium hydrogen phosphate, tartaric acid, acetic acid and succinic acid buffer solution;
the buffer solution B is a sodium bicarbonate buffer solution and/or a borax buffer solution;
the chloride is one or more of magnesium chloride, calcium chloride and lithium chloride;
the stabilizer is one or more of beta-cyclodextrin, ethylene glycol, bovine serum albumin, glycerol and mannitol;
the surfactant is one or more of EMULGEN A-60, EMULGEN A-90, EMULGEN A-500, EMULGEN B-66, EMULGEN LS-114, EMULGEN 109P, EMULGEN123P, EMULGEN 220 and EMULGEN 430;
the preservative is one or more of gentamicin sulfate, sodium azide, 2-hydroxypyridine-N-oxide, chloroacetamide, imidazolidinyl urea, thimerosal, 2-methyl-3 (2H) -isothiazolone, 5-bromo-5-nitro-1, 3-dioxane and Proclin-300.
More preferably, the buffer solution A is 100-200 mmol/L, preferably 100mmol/L, and the pH is 5; the substrate is 1 mmol/L-5 mmol/L, such as 1mmol/L, 3mmol/L, 5 mmol/L; the stabilizer is 1 g/L; the substrate analogues are 1 mmol/L-5 mmol/L, such as 1mmol/L, 3mmol/L, 5 mmol/L; the surfactant accounts for 0.1-1%, preferably 0.5%, and the% accounts for the volume percentage of the reagent R1; the preservative is 1 g/L; the concentration ratio of the substrate approximation object to the substrate is 1: 5-5: 1, preferably 1: 1; the buffer solution B is 100-200 mmol/L, preferably 200mmol/L, and the pH value is 11.
In a preferred embodiment, the reagent R1 comprises: 100mmol/L citric acid buffer solution with pH of 5; and/or, 1mmol/L VRA-NAG; and/or, 1g/L magnesium chloride; and/or, 1g/L EDTA; and/or, 1g/L beta-cyclodextrin; and/or, 1mmol/L of N-acetyl-D-mannosamine, N-acetyl-D-galactosamine or N-acetyl-D-glucosamine; and/or, 1g/L gentamicin sulfate; and/or, EMULGEN a-60 at 0.5%, said% being the volume percentage of said reagent R1;
the reagent R2 comprises: 200mmol/L sodium bicarbonate buffer solution with pH of 11; and/or, 1g/L gentamicin sulfate.
The NAG detection kit preferably further comprises a calibrator, wherein the calibrator comprises a Tris-HCl buffer solution, bovine serum albumin, N-acetyl-beta-D-glucosaminidase and a preservative, and the pH value of the Tris-HCl buffer solution is 7-8.
The calibrator preferably comprises: 10-50 mmol/L Tris-HCl buffer solution, preferably 20mmol/L Tris-HCl buffer solution, and the pH value is 7.0; and/or 10-100U/L of N-acetyl-beta-D-glucosaminidase; and/or 1-10 g/L of bovine serum albumin, preferably 10 g/L; and/or 0.1-5 g/L preservative, preferably 1g/L, such as gentamicin.
In a second aspect, the present invention provides a reagent for detecting NAG, which comprises the reagent R1 in the NAG detection kit according to the first aspect of the present invention.
In a third aspect, the invention provides a method of detecting NAG using a NAG detection kit according to the first aspect of the invention;
preferably:
the dosage ratio of the reagent R1 to the reagent R2 is 3: 1;
the detection is carried out by using a full-automatic protein analyzer, and the wavelength used for the detection is 505nm or 405 nm;
the reaction comprises the following steps:
mixing the reagent R1 with a sample to be detected in a volume ratio of 15:1, and reacting for 3-7 minutes, such as 5 minutes;
and adding the reagent R2, and reacting for 3-7 minutes, such as 5 minutes.
The fourth aspect of the invention provides an application of a substrate approximate analogue in storing and detecting a substrate, wherein the substrate is one or more of VRA-NAG, PNP-NAG and CNP-NAG; the substrate near-analogue is one or more of N-acetyl-D-mannosamine, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine; preferably, the concentration ratio of the substrate mimic to the substrate is 1:5 to 5:1, more preferably 1: 1.
In a fifth aspect, the invention provides a kit for detecting NAG according to the first aspect of the invention or a reagent according to the second aspect of the invention for use in detecting NAG.
The positive progress effects of the invention are as follows:
the kit of the invention uses the substrate analogues for the first time, so that the stability of the reagent is enhanced, particularly the stability of the reagent in the open bottle and the transportation stability are excellent, and the NAG activity in human urine can be rapidly detected. The kit also comprises a liquid calibrator which can be stably stored at 37 ℃ and has good thermal stability.
The kit has good application value for early detection of renal tubular injury, and is the urine enzymology diagnostic item with the highest application rate at present. The kit of the invention uses urine as a specimen, and is a non-invasive test. The kit can be directly used without redissolution, the method is simple and easy to operate, the detection result can be quickly obtained, and the stability is strong, so that the kit is very suitable for clinical large-scale popularization.
Drawings
FIG. 1 is a graph showing the correlation between the reagents for detecting N-acetyl-. beta. -D-glucosaminidase in accordance with the present invention.
Detailed Description
In the following detailed description, numerous specific details are set forth in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps which are well known to those skilled in the art have not been described in detail so as not to obscure the invention.
The experimental principle of the invention is as follows:
the N-acetyl-beta-D-glucosaminidase in urine catalyzes and hydrolyzes 5- [4- (3-methoxy-benzyl-rhodanine) ] -3-ammonium acetate-N-acetamido-beta-D-glucoside (namely VRA-NAG) in the reagent into 5- [4- (3-methoxy-benzyl-rhodanine) ] -3-ammonium acetate (VRA) in an acidic environment, then the VRA is transformed to generate fluorescence in a strong alkaline environment, the solution presents purple red, and has an absorption peak at a wavelength of 505nm, and the activity of the N-acetyl-beta-D-glucosaminidase can be determined by detecting the change of the absorbance at the dominant wavelength of 505 nm.
Wherein VRA-NAG is 5- [4- (3-methoxy-benzyl-rhodanine) ] -3-ammonium acetate-N-acetamido-beta-D-glucoside, and cas number is 125261-87-2; VRA is 5- [4- (3-methoxy-benzyl-rhodanine) ] -3-ammonium acetate; NAG is N-acetyl-beta-D-glucosaminidase.
Similarly, the substrate of the invention can be p-nitrobenzene-N-acetamido-beta-D-glucosaminide (PNP-NAG, cas number 3459-18-5) and 2-chlorine-4-nitrophenyl-N-acetyl-beta-D-glucosaminide (CNP-NAG, cas number 103614-82-0), and when the two substrates are used for detection, the activity of the N-acetyl-beta-D-glucosaminidase can be determined by detecting the change of absorbance at the dominant wavelength of 405 nm.
Example 1
The method for detecting the sample to be detected by using the full-automatic protein analyzer of the biochemical analyzer Hitachi 7180 comprises the following steps: adding 180 mu L of reagent R1 and 12 mu L of sample to be detected into a reaction cup, mixing, incubating at constant temperature of 37 ℃ for 5 minutes, and recording the absorbance A1 under dominant wavelength; an additional 60 μ L of R2 was mixed and after 5 minutes of reaction, the absorbance A2 at the dominant wavelength was recorded and Δ A calculated. The absorbance Δ a1 for the calibrator was also recorded at the dominant wavelength.
Calculating the formula: urine N-acetyl- β -D-glucosaminidase concentration (U/L) ═ sample Δ a ÷ calibrator Δ a1) × calibrator concentration.
As a control, a commercially available and approved urine N-acetyl-beta-D glucosidase assay kit was used, and the components described in the specification were as follows:
r1: MNP-GlcNAc, HCl is more than or equal to 0.1mmol/L, and citric acid (pH 5.0) is more than or equal to 10 mmol/L;
r2: the sodium carbonate buffer solution (pH 11.0) is more than or equal to 100 mmol/L; detecting the dominant wavelength of 505 nm;
calibrator (liquid): the calf serum matrix is more than or equal to 20 percent, and the N-acetyl-beta-D-glucosaminidase is 30-60U/L.
Firstly, the bottle opening stability and the transportation stability of the urine N-acetyl-beta-D glucosidase detection kit which is common and accepted in the market are tested.
The bottle opening stability is as follows: after the reagent is unpacked, the sample is placed in a reagent cabin of a Hitachi 7180 biochemical instrument, the instrument is in a starting state to keep the temperature of a reagent disk, the sample is continuously observed for 7 days, a quality control product (29.8U/L) is measured for 3 times every day, and an average value is taken as a detection result. And observing the change of the detection result of the reagent, and taking the relative deviation exceeding 15% as a judgment standard of runaway. The calculation formula of the relative deviation is as follows:
relative deviation [ (measured value of bottle opening N days C)N0 days measurement of decap C0) 0 day measurement value C0]×100％。
TABLE 1 determination of the stability of this open bottle using quality control (29.8U/L) associated with this kit
| Time to open bottle (Tian) | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Average concentration of quality control product (U/L) | 29.8 | 30.4 | 30.9 | 32.6 | 33.6 | 34.6 | 35.8 | 36.2 |
| Relative deviation of | / | 2％ | 4％ | 9％ | 13％ | 16％ | 20％ | 21％ |
The result shows that the relative deviation of the kit after the kit is opened for 7 days exceeds 20 percent, and the stability of the kit after the kit is opened is poor.
Transportation stability: the kit is respectively mailed to Yunnan and Harbin and then mailed back, and the mailed kit and the kit placed at 2-8 ℃ are used for measuring the quality control product for 3 times.
Relative deviation of [ (measured value of different transport addresses C-2-8 deg.C stored value in refrigerator ℃. ]2～8) Storage measured value C of refrigerator at 2-8 DEG C2～8]×100％
TABLE 2
| Transport address | Preservation in a 2-8 ℃ freezer | Yunnan province | Harbin |
| Average concentration of quality control product (U/L) | 29.6 | 35.3 | 34.4 |
| Relative deviation of | / | 19％ | 16％ |
As a result, as shown in Table 2, the transport stability of the kit was over 15% and it was not stable during transport.
EXAMPLE 2 Effect of different substrates on the decap stability and transport stability of the kits of the invention
The reagent R1 selects 1mmol/L VRA-NAG, PNP-NAG and CNP-NAG as substrates, and other components are as follows: 100mmol/L citrate buffer (pH 5); 1g/L of magnesium chloride; 1g/L of EDTA; 1mmol/L of N-acetyl-D-mannosamine; 1g/L gentamicin sulfate; EMULGEN A-60 at 0.5%; 1g/L beta-cyclodextrin.
R2 component: 200mmol/L sodium bicarbonate buffer (pH 11), 1g/L gentamicin sulfate.
Preparing a calibration product: taking N-acetyl-beta-D-glucosaminidase, dissolving into 10U/L (quality control product 1), 100U/L (quality control product 2) and 50U/L (calibrator) respectively by using 0.02M Tris-HCl solution (pH 7) containing 1% bovine serum albumin, and adding 1g/L gentamicin sulfate.
Determination of the decap stability reference example 1.
TABLE 3 determination of open bottle stability Using Home-made quality control 1(10U/L)
| Time to open bottle (Tian) | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| VRA-NAG substrate (U/L) | 10.1 | 10.0 | 10.3 | 10.5 | 10.2 | 10.4 | 10.5 | 10.6 |
| Relative deviation of | / | -1％ | 2％ | 4％ | 1％ | 3％ | 4％ | 5％ |
| PNP-NAG substrate (U/L) | 10.0 | 9.8 | 10.2 | 10.4 | 10.2 | 10.3 | 10.4 | 10.6 |
| Relative deviation of | / | -2％ | 2％ | 4％ | 2％ | 3％ | 4％ | 6％ |
| CNP-NAG substrate (U/L) | 9.9 | 9.9 | 10.1 | 10 | 10.2 | 10.3 | 10.2 | 10.5 |
| Relative deviation of | / | 0％ | 2％ | 1％ | 3％ | 4％ | 3％ | 6％ |
The result shows that the relative deviation of the bottle opening stability of different substrates is within 10 percent, and the bottle opening stability is good. Indicating that different substrates have little effect on open bottle stability.
The transport stability was determined in reference to example 1.
TABLE 4
| Transport address | Preservation in a 2-8 ℃ freezer | Yunnan province | Harbin |
| VRA-NAG substrate (U/L) | 10.1 | 10.5 | 10.4 |
| Relative deviation of | / | 4％ | 3％ |
| PNP-NAG substrate (U/L) | 9.8 | 10.4 | 10.6 |
| Relative deviation of | / | 6％ | 8％ |
| CNP-NAG substrate (U/L) | 10.2 | 10.6 | 10.8 |
| Relative deviation of | / | 4％ | 6％ |
The results show (see table 4) that the relative deviation of the transportation stability of different substrates and the relative deviation of the kit stored in a refrigerator at 2-8 ℃ are within 10%, and the transportation stability is stable in the transportation process. Indicating that different substrates have little effect on transport stability.
EXAMPLE 3 Effect of different substrate concentrations on decap and Transit stability
The reagent R1 selects 1mmol/L, 3mmol/L and 5mmol/L VRA-NAG as substrate respectively, and other components are as follows: 100mmol/L citric acid buffer (pH: 5); 1g/L of magnesium chloride; 1g/L of EDTA; 1mmol/L of N-acetyl-D-mannosamine; 1g/L gentamicin sulfate; EMULGEN A-60 at 0.5%; 1g/L beta-cyclodextrin.
R2 component: 200mmol/L sodium bicarbonate buffer (pH 11), 1g/L gentamicin sulfate.
Preparing a calibration product: taking N-acetyl-beta-D-glucosaminidase, dissolving into 10U/L (quality control product 1), 100U/L (quality control product 2) and 50U/L (calibrator) respectively by using 0.02M Tris-HCl solution (pH 7) containing 1% bovine serum albumin, and adding 1g/L gentamicin sulfate.
Determination of the decap stability reference example 1.
TABLE 5 decap stability Using self-made quality control 1(10U/L)
| Time to open bottle (Tian) | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| 1mmol/L(U/L) | 10.2 | 10.4 | 10.4 | 10.3 | 10.5 | 10.4 | 10.6 | 10.7 |
| Relative deviation of | / | 2％ | 2％ | 1％ | 3％ | 2％ | 4％ | 5％ |
| 3mmol/L(U/L) | 10.2 | 10.3 | 10.5 | 10.6 | 10.5 | 10.7 | 10.6 | 10.9 |
| Relative deviation of | / | 1％ | 3％ | 4％ | 3％ | 5％ | 4％ | 7％ |
| 5mmol/L(U/L) | 10.4 | 10.8 | 10.9 | 11 | 11.2 | 11.1 | 11.2 | 11.4 |
| Relative deviation of | / | 4％ | 5％ | 6％ | 8％ | 7％ | 8％ | 10％ |
The result shows that the relative deviation of the bottle opening stability of different substrate concentrations is within 10 percent, and the bottle opening stability is good. Indicating that different substrate concentrations had little effect on open vial stability.
The transport stability was determined in reference to example 1.
TABLE 6
The result shows that the relative deviation of the transportation stability of different substrate concentrations and the relative deviation of the kit stored in a refrigerator at 2-8 ℃ are within 10%, and the kit is stable in the transportation process. Indicating that different substrate concentrations have little effect on transport stability.
Example 4 Effect of different substrate analogs on decap and Transit stability
The reagent R1 respectively selects 1mmol/L of N-acetyl-D-mannosamine, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine as substrate analogues and adds no substrate analogues, and other components are as follows: 100mmol/L citrate buffer (pH 5); 1mmol/L VRA-NAG; 1g/L of magnesium chloride; 1g/L of EDTA; 1g/L gentamicin sulfate; EMULGEN A-60 at 0.5%; 1g/L beta-cyclodextrin.
R2 component: 200mmol/L sodium bicarbonate buffer (pH 11), 1g/L gentamicin sulfate.
Preparing a calibration product: taking N-acetyl-beta-D-glucosaminidase, dissolving into 10U/L (quality control product 1), 100U/L (quality control product 2) and 50U/L (calibrator) respectively by using 0.02M Tris-HCl solution (pH 7) containing 1% bovine serum albumin, and adding 1g/L gentamicin sulfate.
Determination of the decap stability reference example 1.
TABLE 7 open bottle stability measured using Home-made quality control 1(10U/L)
| Time to open bottle (Tian) | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| N-acetyl-D-mannosamine (U/L) | 10.3 | 10.2 | 10.3 | 10.5 | 10.6 | 10.4 | 10.7 | 10.6 |
| Relative deviation of | / | -1％ | 0％ | 2％ | 3％ | 1％ | 4％ | 3％ |
| N-acetyl-D-galactosamine (U/L) | 10.2 | 10.3 | 10.2 | 10.3 | 10.4 | 10.6 | 10.5 | 10.8 |
| Relative deviation of | / | 1％ | 0％ | 1％ | 2％ | 4％ | 3％ | 6％ |
| N-acetyl-D-glucosamine (U/L) | 9.9 | 10 | 10.1 | 10.2 | 10.4 | 10.5 | 10.4 | 10.7 |
| Relative deviation of | / | 1％ | 2％ | 3％ | 5％ | 6％ | 5％ | 8％ |
| Without substrate approximation (U/L) | 10.2 | 10.5 | 10.8 | 11.2 | 11.5 | 11.8 | 12.1 | 12.4 |
| Relative deviation of | / | 3％ | 6％ | 10％ | 13％ | 16％ | 19％ | 22％ |
The result shows that the relative deviation of the bottle opening stability of different substrate analogues is within 10 percent, and the bottle opening stability is good; the relative variation of the open bottle of the non-substrate-added approximation-object for 7 days was over 20%, which is poor in open bottle stability, indicating that different substrate approximation-objects have little effect on open bottle stability, but the reagent without substrate approximation-added approximation-object has poor open bottle stability.
The transport stability was determined in reference to example 1.
TABLE 8
| Transport address | Preservation in a 2-8 ℃ freezer | Yunnan province | Harbin |
| N-acetyl-D-mannosamine (U/L) | 10.2 | 10.6 | 10.7 |
| Relative deviation of | / | 4％ | 5％ |
| N-acetyl-D-galactosamine (U/L) | 10.3 | 10.8 | 10.9 |
| Relative deviation of | / | 5％ | 6％ |
| Without substrate approximation (U/L) | 10.1 | 11.8 | 11.7 |
| Relative deviation of | / | 17％ | 16％ |
The results show that the relative deviation of the transport stability of different substrate analogues and the relative deviation of the kit stored in a refrigerator at 2-8 ℃ are within 10%, the transport stability of the substrate analogues is stable in the transport process, and the relative deviation of the transport stability of the substrate analogues not added and the relative deviation of the kit stored in the refrigerator at 2-8 ℃ is over 15%. It was shown that different substrate mimetics had little effect on trafficking stability, but that the trafficking stability was poor without the addition of substrate mimetics.
Example 5 Effect of different substrate approximation concentrations on decap and Transit stability
The reagent R1 is N-acetyl-D-mannosamine with the concentration of 1mmol/L, 3mmol/L and 5mmol/L respectively, and other components are as follows: 100mmol/L citrate buffer (pH 5); 1mmol/L VRA-NAG; 1g/L of magnesium chloride; 1g/L of EDTA; 1g/L gentamicin sulfate; EMULGEN A-60 at 0.5%; 1g/L beta-cyclodextrin.
R2 component: 200mmol/L sodium bicarbonate buffer (pH 11), 1g/L gentamicin sulfate.
Preparing a calibration product: taking N-acetyl-beta-D-glucosaminidase, dissolving into 10U/L (quality control product 1), 100U/L (quality control product 2) and 50U/L (calibrator) respectively by using 0.02M Tris-HCl solution (pH 7) containing 1% bovine serum albumin, and adding 1g/L gentamicin sulfate.
Determination of the decap stability reference example 1.
TABLE 9 decap stability Using Home-made quality control 1(10U/L)
The results show that the relative deviation of the bottle opening stability of different substrate approximate compound concentrations is within 10 percent, and the bottle opening stability is good. Indicating that different substrate mimic concentrations had little effect on open vial stability.
The transport stability was determined in reference to example 1.
| Transport address | Preservation in a 2-8 ℃ freezer | Yunnan province | Harbin |
| 1mmol/L(U/L) | 10.2 | 10.6 | 10.8 |
| Relative deviation of | / | 4％ | 6％ |
| 3mmol/L(U/L) | 10.3 | 11.2 | 11 |
| Relative deviation of | / | 9％ | 7％ |
The results show that the relative deviation of the transportation stability of different substrate near-analogue concentrations and the relative deviation of the kit stored in a refrigerator at 2-8 degrees are within 10 percent, and the different substrate near-analogue concentrations are stable in the transportation process, and show that the transportation stability is not greatly influenced by the different substrate near-analogue concentrations.
EXAMPLE 6 Effect of different chlorides on decap and Transit stability
The reagent R1 is prepared from 1g/L of magnesium chloride, calcium chloride and lithium chloride respectively, and other components: 100mmol/L citrate buffer (pH 5); 1mmol/L VRA-NAG; 1g/L of EDTA; 1mmol/L of N-acetyl-D-mannosamine; 1g/L gentamicin sulfate; EMULGEN A-60 at 0.5%; 1g/L beta-cyclodextrin.
R2 component: 200mmol/L sodium bicarbonate buffer (pH 11), 1g/L gentamicin sulfate.
Preparing a calibration product: taking N-acetyl-beta-D-glucosaminidase, dissolving into 10U/L (quality control product 1), 100U/L (quality control product 2) and 50U/L (calibrator) respectively by using 0.02M Tris-HCl solution (pH 7) containing 1% bovine serum albumin, and adding 1g/L gentamicin sulfate.
Determination of the decap stability reference example 1.
TABLE 11 decap stability Using Home-made quality control 1(10U/L)
The result shows that the relative deviation of the bottle opening stability of different chlorides is within 10 percent, and the bottle opening stability is good. Indicating that different chlorides have little effect on the open bottle stability.
The transport stability was determined in reference to example 1.
TABLE 12
| Time to open bottle (Tian) | Preservation in a 2-8 ℃ freezer | Yunnan province | Harbin |
| Magnesium chloride (U/L) | 10.2 | 10.8 | 10.9 |
| Relative deviation of | / | 6％ | 7％ |
| Calcium chloride (U/L) | 10.3 | 11 | 11.2 |
| Relative deviation of | / | 7％ | 9％ |
| Lithium chloride (U/L) | 10 | 10.8 | 10.9 |
| Relative deviation of | / | 8％ | 9％ |
The results show that the relative deviation between the transportation stability of different chlorides and the kit stored in the refrigerator at the temperature of 2-8 ℃ are within 10 percent, and the transportation stability is stable in the transportation process, which shows that the transportation stability is not greatly influenced by different chlorides.
Example 7 Effect of different surfactants on bottle opening stability and transportation stability
The reagent R1 selects 0.5% of EMULGEN A-60, EMULGEN A-90 and EMULGEN A-500 as the surfactant respectively, and other components are as follows: 100mmol/L citrate buffer (pH 5); 1mmol/L VRA-NAG; 1g/L of magnesium chloride; 1g/L of EDTA; 1mmol/L of N-acetyl-D-mannosamine; 1g/L gentamicin sulfate; 1g/L beta-cyclodextrin.
R2 component: 200mmol/L sodium bicarbonate buffer (pH 11), 1g/L gentamicin sulfate.
Preparing a calibration product: taking N-acetyl-beta-D-glucosaminidase, dissolving into 10U/L (quality control product 1), 100U/L (quality control product 2) and 50U/L (calibrator) respectively by using 0.02M Tris-HCl solution (pH 7) containing 1% bovine serum albumin, and adding 1g/L gentamicin sulfate.
Determination of the decap stability reference example 1.
TABLE 13 bottle opening stability Using Home-made quality control 1(10U/L)
The result shows that the relative deviation of the bottle opening stability of different surfactants is within 10 percent, and the bottle opening stability is good. Indicating that different surfactants have little effect on the open bottle stability.
The transport stability was determined in reference to example 1.
TABLE 14
| Time to open bottle (Tian) | Preservation in a 2-8 ℃ freezer | Yunnan province | Harbin |
| EMULGEN A-60(U/L) | 10.1 | 10.7 | 10.9 |
| Relative deviation of | / | 6％ | 8％ |
| EMULGEN A-90(U/L) | 10.2 | 11.1 | 11.2 |
| Relative deviation of | / | 9％ | 10％ |
| EMULGEN A-500(U/L) | 10 | 10.9 | 10.9 |
| Relative deviation of | / | 9％ | 9％ |
The results show that the relative deviation between the transportation stability of different surfactants and the kit stored in a refrigerator at 2-8 ℃ are within 10%, and the transportation stability is stable in the transportation process, which shows that the transportation stability is not greatly influenced by different surfactants.
EXAMPLE 8 Effect of different stabilizers on bottle opening stability and transportation stability
The reagent R1 selects 1g/L beta-cyclodextrin, glycol and bovine serum albumin as stabilizers respectively, and other components are as follows: 100mmol/L citrate buffer (pH 5); 1mmol/L VRA-NAG; 1g/L of magnesium chloride; 1g/L of EDTA; 1mmol/L of N-acetyl-D-mannosamine; 1g/L gentamicin sulfate; EMULGEN A-60 of 0.5%.
R2 component: 200mmol/L sodium bicarbonate buffer (pH 11), 1g/L gentamicin sulfate.
Preparing a calibration product: taking N-acetyl-beta-D-glucosaminidase, dissolving into 10U/L (quality control product 1), 100U/L (quality control product 2) and 50U/L (calibrator) respectively by using 0.02M Tris-HCl solution (pH 7) containing 1% bovine serum albumin, and adding 1g/L gentamicin sulfate.
Determination of the decap stability reference example 1.
TABLE 15 decap stability Using Home-made quality control 1(10U/L)
The result shows that the relative deviation of the bottle opening stability of different stabilizers is within 10 percent, and the bottle opening stability is good. Indicating that different stabilizers have little effect on the open bottle stability.
The transport stability was determined in reference to example 1.
TABLE 16
| Time to open bottle (Tian) | Preservation in a 2-8 ℃ freezer | Yunnan province | Harbin |
| Beta-cyclodextrin (U/L) | 10.1 | 10.7 | 10.7 |
| Relative deviation of | / | 6％ | 6％ |
| Ethylene glycol (U/L) | 10.2 | 11.0 | 10.8 |
| Relative deviation of | / | 8％ | 6％ |
| Bovine serum albumin (U/L) | 10 | 10.7 | 10.8 |
| Relative deviation of | / | 7％ | 8％ |
The results show that the relative deviation between the transportation stability of different stabilizers and the relative deviation between the different stabilizers and the kit stored in a refrigerator with the temperature of 2-8 ℃ are within 10 percent, and the different stabilizers are stable in the transportation process, which shows that the different stabilizers have little influence on the transportation stability.
EXAMPLE 9 Effect of different preservatives on bottle opening stability and transportation stability
The reagent R1 respectively selects 1g/L gentamicin sulfate, sodium azide and Proclin-300 as preservatives, and other components are as follows: 100mmol/L citrate buffer (pH 5); 1mmol/L VRA-NAG; 1g/L of magnesium chloride; 1g/L of EDTA; 1mmol/L of N-acetyl-D-mannosamine; 1g/L beta-cyclodextrin; EMULGEN A-60 at 0.5%; 1g/L beta-cyclodextrin.
R2 component: 200mmol/L sodium bicarbonate buffer (pH 11), 1g/L gentamicin sulfate.
Preparing a calibration product: taking N-acetyl-beta-D-glucosaminidase, dissolving into 10U/L (quality control product 1), 100U/L (quality control product 2) and 50U/L (calibrator) respectively by using 0.02M Tris-HCl solution (pH 7) containing 1% bovine serum albumin, and adding 1g/L gentamicin sulfate.
Determination of the decap stability reference example 1.
TABLE 17 bottle opening stability Using Home-made quality control 1(10U/L)
The result shows that the relative deviation of the bottle opening stability of different preservatives is within 10 percent, and the bottle opening stability is good. Indicating that different preservatives have little effect on the open bottle stability.
The transport stability was determined in reference to example 1.
Watch 18
| Time to open bottle (Tian) | Preservation in a 2-8 ℃ freezer | Yunnan province | Harbin |
| Gentamicin sulfate (U/L) | 10.1 | 10.7 | 10.7 |
| Relative deviation of | / | 6％ | 6％ |
| Sodium azide (U/L) | 10.2 | 11.0 | 10.8 |
| Relative deviation of | / | 8％ | 6％ |
| Proclin-300(U/L) | 10 | 10.7 | 10.8 |
| Relative deviation of | / | 7％ | 8％ |
The results show that the relative deviation between the transportation stability of different preservatives and the relative deviation between the different preservatives and the kit stored in the refrigerator with the temperature of 2-8 ℃ are within 10 percent, and the different preservatives are stable in the transportation process, which shows that the different preservatives have little influence on the transportation stability.
EXAMPLE 10 Effect of different R1 buffers on decap and Transit stability
Reagent R1 selects 100mmol/L citric acid buffer solution (pH 5), 100mmol/L citric acid-disodium hydrogen phosphate (pH 5), 100mmol/L tartaric acid buffer solution (pH 5), and other components: 1mmol/L VRA-NAG; 1g/L of magnesium chloride; 1g/L of EDTA; 1mmol/L of N-acetyl-D-mannosamine; 1g/L gentamicin sulfate; EMULGEN A-60 at 0.5%; 1g/L beta-cyclodextrin.
R2 component: 200mmol/L sodium bicarbonate buffer (pH 11), 1g/L gentamicin sulfate.
Preparing a calibration product: taking N-acetyl-beta-D-glucosaminidase, dissolving into 10U/L (quality control product 1), 100U/L (quality control product 2) and 50U/L (calibrator) respectively by using 0.02M Tris-HCl solution (pH 7) containing 1% bovine serum albumin, and adding 1g/L gentamicin sulfate.
Determination of the decap stability reference example 1.
TABLE 19 decap stability Using self-made quality control 1(10U/L)
The results show that the relative deviation of the bottle opening stability of different R1 buffer solutions is within 10 percent, and the bottle opening stability is good. Indicating that different R1 buffers had little effect on open vial stability.
The transport stability was determined in reference to example 1.
| Time to open bottle (Tian) | Preservation in a 2-8 ℃ freezer | Yunnan province | Harbin |
| Citric acid buffer (U/L) | 10 | 10.8 | 10.6 |
| Relative deviation of | / | 8％ | 6％ |
| Citric acid-disodium hydrogen phosphate | 10.1 | 10.9 | 10.7 |
| Relative deviation (U/L) | / | 8％ | 6％ |
| Tartaric acid buffer solution (U/L) | 10 | 10.9 | 10.8 |
| Relative deviation of | / | 9％ | 8％ |
The results show that the relative deviation between the transport stability of different R1 buffer solutions and the relative deviation between the transport stability and the kit stored in a refrigerator at 2-8 ℃ are within 10%, and the transport stability is stable in the transport process, which indicates that the transport stability is not greatly influenced by different R1 buffer solutions.
EXAMPLE 11 Effect of different R2 buffers on decap and Transit stability
Reagent R1: 100mmol/L citrate buffer (pH 5); 1mmol/L VRA-NAG; 1g/L of magnesium chloride; 1g/L of EDTA; 1mmol/L of N-acetyl-D-mannosamine; 1g/L gentamicin sulfate; EMULGEN A-60 at 0.5%; 1g/L beta-cyclodextrin.
R2 respectively selecting 200mmol/L sodium bicarbonate buffer solution (pH 11) and 200mmol/L borax buffer solution (pH 11); and 1g/L gentamicin sulfate is added.
Preparing a calibration product: taking N-acetyl-beta-D-glucosaminidase, dissolving into 10U/L (quality control product 1), 100U/L (quality control product 2) and 50U/L (calibrator) respectively by using 0.02M Tris-HCl solution (pH 7) containing 1% bovine serum albumin, and adding 1g/L gentamicin sulfate.
Determination of the decap stability reference example 1.
TABLE 21 open bottle stability determined using Home-made quality control 1(10U/L)
| Time to open bottle (Tian) | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Sodium bicarbonate buffer (U/L) | 10.1 | 10.2 | 10.3 | 10.4 | 10.5 | 10.6 | 10.7 | 10.9 |
| Relative deviation of | / | 1％ | 2％ | 3％ | 4％ | 5％ | 6％ | 8％ |
| Borax buffer (U/L) | 10.0 | 10.1 | 10.2 | 10.3 | 10.5 | 10.5 | 10.7 | 10.8 |
| Relative deviation (U/L) | / | 1％ | 2％ | 3％ | 5％ | 5％ | 7％ | 8％ |
The results show that the relative deviation of the bottle opening stability of different R2 buffer solutions is within 10 percent, and the bottle opening stability is good. Indicating that different R2 buffers had little effect on open vial stability.
Table 22 determination of transport stability reference example 1.
| Time to open bottle (Tian) | Preservation in a 2-8 ℃ freezer | Yunnan province | Harbin |
| Sodium bicarbonate buffer (U/L) | 10 | 10.8 | 10.6 |
| Relative deviation of | / | 8％ | 6％ |
| Borax buffer (U/L) | 10.1 | 10.9 | 10.7 |
| Relative deviation (U/L) | / | 8％ | 6％ |
The results show that the relative deviation between the transport stability of different R2 buffer solutions and the relative deviation between the transport stability and the kit stored in a refrigerator at 2-8 ℃ are within 10%, and the transport stability is stable in the transport process, which indicates that the transport stability is not greatly influenced by different R2 buffer solutions.
EXAMPLE 12 reagent and calibrator thermal stability test
Thermal stability of the reagent: the quality control product is tested 3 times by using the reagents R1 and R2, the reagents R1 and R2 are placed in a constant temperature incubator at 37 ℃ for 7 days, and then the quality control product is tested 3 times by using the reagents R1 and R2 placed in the constant temperature incubator at 37 ℃ for 7 days.
Thermal stability of the calibrator: the quality control product is tested 3 times by using the reagents R1 and R2, the quality control product is placed in a constant temperature incubator at 37 ℃ for 7 days, and then the quality control product is tested 3 times by using the reagents R1 and R2 after being placed in the constant temperature incubator at 37 ℃ for 7 days.
Reagent R1: 100mmol/L citrate buffer (pH 5); 1mmol/L VRA-NAG; 1g/L of magnesium chloride; 1g/L of EDTA; 1mmol/L of N-acetyl-D-mannosamine; 1g/L gentamicin sulfate; EMULGEN A-60 at 0.5%; 1g/L beta-cyclodextrin.
Reagent R2: 200mmol/L sodium bicarbonate buffer (pH 11), 1g/L gentamicin sulfate.
Preparing a calibration product: taking N-acetyl-beta-D-glucosaminidase, dissolving into 10U/L (quality control product 1), 100U/L (quality control product 2) and 50U/L (calibrator) respectively by using 0.02M Tris-HCl solution (pH 7) containing 1% bovine serum albumin, and adding 1g/L gentamicin sulfate.
The calculation formula of the relative deviation is as follows:
relative deviation [ (measured after placing in a 37 ℃ incubator for 7 days ] measured before placing in a 37 ℃ incubator for C-0) Before placing in a constant temperature incubator at 37 ℃, measuring value C0]×100％
TABLE 23 thermal stability of reagents
| Before being put into a constant temperature incubator at 37 DEG C | Placing in a constant temperature incubator at 37 ℃ for 7 days | Relative deviation of | |
| Quality control product 1(U/L) | 10.1 | 10.8 | 7％ |
| Quality control product 2(U/L) | 100.3 | 105.2 | 5％ |
TABLE 24 thermal stability of calibrators
| Before being put into a constant temperature incubator at 37 DEG C | Placing in a constant temperature incubator at 37 ℃ for 7 days | Relative deviation of | |
| Quality control product 1(U/L) | 10.1 | 10.5 | 4％ |
| Quality control 2(U/L)) | 100.3 | 103.5 | 3％ |
The result shows that the kit calibrator of the kit has better thermal stability.
Example 13 correlation experiment
The reagent formula of the embodiment is used for preparing a detection reagent, and the detection reagent is compared with a common N-acetyl-beta-D glucosidase detection kit in the market for detection, and 50 clinical urine samples are respectively detected in each embodiment group.
The kit comprises the following components:
reagent R1: 100mmol/L citrate buffer (pH 5); 1mmol/L VRA-NAG; 1g/L of magnesium chloride; 1g/L of EDTA; 1mmol/L of N-acetyl-D-mannosamine; 1g/L gentamicin sulfate; EMULGEN A-60 at 0.5%; 1g/L beta-cyclodextrin.
Reagent R2: 200mmol/L sodium bicarbonate buffer (pH 11), 1g/L gentamicin sulfate.
The reagent for detecting the N-acetyl-beta-D glucosidase in urine, which is commonly and approved in the market, comprises the following components in the specification:
r1: MNP-GlcNAc, HCl not less than 0.1mmol/L, citric acid (pH 5.0) not less than 10 mmol/L;
r2: the sodium carbonate buffer solution (pH 11.0) is more than or equal to 100 mmol/L.
The results are given in Table 25 below:
TABLE 25 correlation determination
As can be seen from FIG. 1, the kit has good correlation.
Claims (10)
1. An NAG detection kit, which is characterized by comprising a reagent R1 and a reagent R2;
the reagent R1 comprises:
10-500 mmol/L buffer solution A, pH 4-6; 0.1-5 mmol/L of substrate; 1-5 g/L chloride; 1-5 g/L of EDTA or sodium salt thereof; 0.1-5 g/L stabilizer; 0.1-5 mmol/L of substrate approximation compound; 0.1-5% of surfactant, wherein the% is the volume percentage of the reagent R1; 0.1-5 g/L of preservative; the substrate is one or more of VRA-NAG, PNP-NAG and CNP-NAG;
the reagent R2 comprises: 10-500 mmol/L buffer solution B with the pH value of 10-12; 0.1-5 g/L of preservative;
preferably, the substrate mimic is one or more of N-acetyl-D-mannosamine, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine.
2. The NAG detection kit of claim 1, wherein the buffer a is one or more of a citric acid buffer, potassium hydrogen phthalate, citric acid-disodium hydrogen phosphate, tartaric acid, acetic acid and succinic acid buffer;
and/or the buffer solution B is a sodium bicarbonate buffer solution and/or a borax buffer solution;
and/or the chloride is one or more of magnesium chloride, calcium chloride and lithium chloride;
and/or the stabilizer is one or more of beta-cyclodextrin, ethylene glycol, bovine serum albumin, glycerol and mannitol;
and/or the surfactant is one or more of EMULGEN a-60, EMULGEN a-90, EMULGEN a-500, EMULGEN B-66, EMULGEN LS-114, EMULGEN 109P, EMULGEN123P, EMULGEN 220, and EMULGEN 430;
and/or the preservative is one or more of gentamicin sulfate, sodium azide, 2-hydroxypyridine-N-oxide, chloroacetamide, imidazolidinyl urea, thimerosal, 2-methyl-3 (2H) -isothiazolone, 5-bromo-5-nitro-1, 3-dioxane and Proclin-300.
3. The NAG detection kit of claim 1 or 2, wherein the buffer solution A is 100 to 200mmol/L, preferably 100mmol/L, and the pH is 5; and/or the substrate is 1 mmol/L-5 mmol/L; and/or the stabilizer is 1 g/L; and/or the substrate approximation object is 1 mmol/L-5 mmol/L; and/or, the surfactant is 0.1% to 1%, preferably 0.5%, the% being in volume percentage of the reagent R1; and/or the preservative is 1 g/L; and/or the concentration ratio of the substrate approximation object to the substrate is 1: 5-5: 1, preferably 1: 1; and/or the buffer solution B is 100-200 mmol/L, preferably 200mmol/L, and the pH value is 11.
4. The NAG detection kit according to any one of claims 1 to 3, wherein the reagent R1 comprises: 100mmol/L citric acid buffer solution with pH of 5; and/or, 1mmol/L VRA-NAG; and/or, 1g/L magnesium chloride; and/or, 1g/L EDTA; and/or, 1g/L beta-cyclodextrin; and/or, 1mmol/L of N-acetyl-D-mannosamine, N-acetyl-D-galactosamine or N-acetyl-D-glucosamine; and/or, 1g/L gentamicin sulfate; and/or, EMULGEN a-60 at 0.5%, said% being the volume percentage of said reagent R1;
the reagent R2 comprises: 200mmol/L sodium bicarbonate buffer solution with pH of 11; and/or, 1g/L gentamicin sulfate.
5. The NAG detection kit of any one of claims 1 to 4, further comprising a calibrator comprising Tris-HCl buffer, bovine serum albumin, N-acetyl- β -D-glucosaminidase, and a preservative, wherein the pH of the Tris-HCl buffer is 7 to 8.
6. The NAG detection kit of claim 5, wherein the calibrator comprises: 10-50 mmol/L Tris-HCl buffer solution, preferably 20mmol/L, pH is 7.0; and/or 10-100U/L of N-acetyl-beta-D-glucosaminidase; and/or 1-10 g/L of bovine serum albumin, preferably 10 g/L; and/or 0.1-5 g/L, preferably 1g/L, of a preservative, such as gentamicin.
7. A reagent for detecting NAG, which comprises the reagent R1 in the NAG detection kit according to any one of claims 1 to 6.
8. A method for detecting NAG, characterized in that the detection is carried out by using the NAG detection kit according to any one of claims 1 to 6;
preferably:
the dosage ratio of the reagent R1 to the reagent R2 is 3: 1;
and/or the detection is carried out by using a full-automatic protein analyzer, and the wavelength used for the detection is 505nm or 405 nm;
and/or, the reaction comprises the steps of:
mixing the reagent R1 with a sample to be detected in a volume ratio of 15:1, and reacting for 3-7 minutes, such as 5 minutes;
and adding the reagent R2, and reacting for 3-7 minutes, such as 5 minutes.
9. The application of a substrate near-analog in substrate preservation and detection is characterized in that the substrate is one or more of VRA-NAG, PNP-NAG and CNP-NAG; the substrate near-analogue is one or more of N-acetyl-D-mannosamine, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine; preferably, the concentration ratio of the substrate mimic to the substrate is 1:5 to 5:1, more preferably 1: 1.
10. Use of the NAG detection kit according to any one of claims 1 to 6 or the reagent according to claim 7 for the detection of NAG.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6252000A (en) * | 1985-08-30 | 1987-03-06 | Toyobo Co Ltd | Reagent for measuring beta-n-acetyl-d-hexosaminidase activity |
| CN101738379A (en) * | 2009-12-31 | 2010-06-16 | 宁波美康生物科技有限公司 | Liquid reagent for determining N-acetyl-beta-D-glucosaminidase |
| CN112501245A (en) * | 2020-11-09 | 2021-03-16 | 山东博科生物产业有限公司 | Novel N-acetyl-beta-D glucosaminidase detection reagent |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6252000A (en) * | 1985-08-30 | 1987-03-06 | Toyobo Co Ltd | Reagent for measuring beta-n-acetyl-d-hexosaminidase activity |
| CN101738379A (en) * | 2009-12-31 | 2010-06-16 | 宁波美康生物科技有限公司 | Liquid reagent for determining N-acetyl-beta-D-glucosaminidase |
| CN112501245A (en) * | 2020-11-09 | 2021-03-16 | 山东博科生物产业有限公司 | Novel N-acetyl-beta-D glucosaminidase detection reagent |
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| Title |
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| 陈晓婷等: "尿氨基己糖苷酶对乙醇性肝病的诊断", 《江苏大学学报(医学版)》 * |
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