Creatinine detection kit with good stability and strong interference resistance

Abstract

The invention relates to the technical field of biochemical detection, in particular to a creatinine detection kit with good stability and strong interference resistance, the creatinine detection kit comprises a reagent R1 and a reagent R2, the reagent R1 comprises a buffer solution, a bacteriostatic agent, a surfactant, an enzyme activator, chromogen, creatinase, sarcosine oxidase and peroxidase, and the reagent R2 comprises a buffer solution, a preservative, a color developing agent and creatinase; the creatininase method detection kit provided by the invention can realize simultaneous detection of blood and urine, and is good in stability and strong in anti-interference capability; by adding the bacteriostatic agent, the stability of the kit is greatly improved; by adding the surfactant, the interference of a chyle sample on the kit is improved, the anti-interference capability is strong, and the detection accuracy of the kit is improved; the linear range of the kit is increased by adding the enzyme activator, and the detection performance of the kit is optimized.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical detection, and particularly relates to a creatinine detection kit with good stability and strong anti-interference ability. Background Art

[0002] Creatinine (Cr) is the end product of human muscle metabolism, mainly formed by the irreversible non-enzymatic dehydration reaction of creatine, released into the blood, excreted in urine, not affected by diet and exercise, etc. It is only filtered through the glomerulus and not reabsorbed by the renal tubules, so it can accurately reflect the glomerular filtration rate function. At present, the clinical detection of creatinine includes serum creatinine and urine creatinine detection, and the determination usually uses the enzymatic method, among which the sarcosine oxidase method is the most common. The reaction principle of the sarcosine oxidase method: Creatinine is hydrolyzed to form creatine under the catalysis of creatinine enzyme, and then creatine is hydrolyzed to produce sarcosine and urea under the catalysis of creatine enzyme. Sarcosine is oxidized to glycine, formaldehyde and hydrogen peroxide under the catalysis of sarcosine oxidase, and finally the quinoneimine pigment formed by coupling the Trinder reaction is proportional to the concentration of creatinine, and the colorimetric method is used for determination.

[0003] However, the sarcosine oxidase method has the following problems:

[0004] The creatinine detection kit is affected by the linear range and cannot meet the need for simultaneous detection of creatinine in blood and urine; there are many tool enzymes such as creatine enzyme, creatinine enzyme, sarcosine oxidase, etc. in the above method, which have an adverse effect on the stability of the creatinine detection kit; when the Trinder reaction develops color, the lipids in the chylous sample will increase the turbidity of the sample, resulting in light scattering or absorption interference during the absorbance measurement, affecting the detection accuracy. Summary of the Invention

[0005] Aiming at the technical problems in the prior art, the purpose of the present invention is to provide a creatinine detection kit with good stability and strong anti-interference ability, which can realize the simultaneous detection of blood and urine, and has strong stability and anti-interference ability.

[0006] To achieve the above purpose, the technical solution provided by the present invention is as follows:

[0007] The present invention provides a creatinine detection kit with good stability and strong anti-interference ability, comprising the following components: reagent R1 and reagent R2; wherein,

[0008] Reagent R1 includes buffer 10 - 20 g / L, bacteriostatic agent 0.1 - 0.5 g / L, surfactant 0.2 - 0.5 g / L, enzyme activator 1 - 10 g / L, chromogen 0.5 - 1.5 g / L, creatine enzyme 50 - 150 KU / L, sarcosine oxidase 10 - 20 KU / L and peroxidase 1 - 10 KU / L;

[0009] Reagent R2 includes 10 - 20 g / L of buffer, 1 - 5 g / L of preservative, 0.5 - 1.5 g / L of chromogenic agent, and 150 - 400 KU / L of creatinine enzyme.

[0010] The present invention provides a creatinine enzyme method detection kit, which can realize the simultaneous detection of blood and urine, has good stability and strong anti-interference ability. Specifically, by adding bacteriostatic agents, the stability of the kit is greatly improved; by adding surfactants, the interference of chylous samples to the kit is improved, the anti-interference ability is strong, and the detection accuracy of the kit is improved; by adding enzyme activators, the linear range of the kit is increased, and the detection performance of the kit is optimized.

[0011] Based on the above technical solution, the bacteriostatic agent in reagent R1 is gentamicin.

[0012] Specifically, using gentamicin as the bacteriostatic agent to enhance the stability of the kit, its mechanism of action is: gentamicin can bind to bacterial ribosomes, affect the process of bacterial protein synthesis, inhibit the growth and reproduction of bacteria, and even destroy the integrity of the bacterial cell wall, resulting in the death of bacteria, reducing the risk of bacterial contamination of the kit, thereby enhancing the stability of the kit.

[0013] Based on the above technical solution, the surfactant of reagent R1 is one or two of fatty alcohol polyoxyethylene ether AEO-7 and AEO-9.

[0014] The above AEO-7 refers to fatty alcohol polyoxyethylene ether compounds, 7 refers to the polymerization of fatty alcohol with 7 ethylene oxide units, and AEO-9 uses 9 ethylene oxide units.

[0015] By using fatty alcohol polyoxyethylene ether compounds as surfactants, the interference of chylous samples to the kit is improved, and the detection accuracy of the kit is improved. Its mechanism of action is: surfactants AEO-7 and AEO-9, on the one hand, can significantly reduce the surface tension of the reagent and weaken the interaction between chylomicrons; on the other hand, through solubilization, the distribution of chylomicrons in the sample is more uniform, reducing the scattering light effect caused by the aggregation of chylomicrons. At the same time, the surfactant can also stabilize the enzyme protein structure, reduce non-specific adsorption, and cooperate with the enzyme activator. It can not only accelerate a series of enzymatic hydrolysis reactions during the detection of creatinine reagent, but also shield the interference of poor accuracy of the kit caused by too many tool enzymes, further improving the detection accuracy of the kit.

[0016] Based on the above technical solution, the enzyme activator of reagent R1 is one or two of KCl and NaCl.

[0017] By using potassium chloride and sodium chloride as enzyme activators, the linear range of the kit is increased and the detection performance of the kit is optimized. The mechanism of action is as follows: The potassium ions K+ and sodium ions Na+ in KCl and NaCl, on the one hand, combine with the enzyme to form a complex, stabilize the conformation of the enzyme molecule, and enhance the catalytic action of the enzyme; on the other hand, they promote the binding of the substrate to the enzyme, thereby accelerating the reaction rate and playing an activating role. The problem that the poor stability of the kit components caused by the large number of tool enzymes affects the linear range of the kit is solved. In addition, when the enzyme activator and the bacteriostatic agent are used simultaneously, the bacteriostatic agent binds to the metal ions in the enzyme activator, which not only greatly enhances the effectiveness of the enzyme activator as a cofactor, but also is conducive to the stability of the reaction system.

[0018] On the basis of the above technical solution, the chromogen of reagent R1 is sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline TOOS.

[0019] On the basis of the above technical solution, the preservative of reagent R2 is NaN3.

[0020] On the basis of the above technical solution, the chromogenic agent of reagent R2 is 4-aminoantipyrine.

[0021] On the basis of the above technical solution, the buffer solutions of reagent R1 and reagent R2 are both 3-(N-morpholino)-2-hydroxypropanesulfonic acid MOPSO buffer solution and the pH range is 6.5 - 8.5.

[0022] On the basis of the above technical solution, the volume ratio of reagent R1 to reagent R2 is 3:1.

[0023] The beneficial effects produced by the technical solution provided by the present invention are as follows:

[0024] The present invention provides a kit for detecting sarcosine oxidase method, which can realize the simultaneous detection of blood and urine, has good stability and strong anti-interference ability. Specifically, by adding a bacteriostatic agent, the stability of the kit is greatly improved; by adding a surfactant, the interference of chylous samples on the kit is improved, the anti-interference ability is strong, and the detection accuracy of the kit is improved; by adding an enzyme activator, the linear range of the kit is increased and the detection performance of the kit is optimized. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a result characterization diagram of the bacteriostatic experiment; among them, (a) is a commercially available kit, and (b)-(d) are the kits prepared in Examples 1-3 respectively;

[0026] Figure 2 It is a linear correlation performance characterization diagram; among them, (a)-(c) are the kits prepared in Examples 1-3 respectively; DETAILED DESCRIPTION OF THE INVENTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the content in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in this specification includes any and all combinations of one or more of the related listed items. It should be understood that unless otherwise specified, various raw materials in the present invention can be obtained commercially.

[0029] Example 1

[0030] The components of the sarcosine oxidase method detection kit in Example 1 are as follows:

[0031]

[0032] The preparation methods of reagent R1 and reagent R2 are conventional methods, that is, the components of reagent R1 and reagent R2 are respectively added to purified water and then mixed and stirred evenly; among them, the pH value of the MOPSO buffer solution is 7.5.

[0033] Example 2

[0034] The components of the sarcosine oxidase method detection kit in Example 2 are as follows:

[0035]

[0036] Among them, the preparation method of Example 2 is the same as that of Example 1.

[0037] Example 3

[0038] The components of the sarcosine oxidase method detection kit in Example 3 are as follows:

[0039]

[0040] Among them, the preparation method of Example 3 is the same as that of Example 1.

[0041] Experimental results and performance analysis

[0042] The kit of the present application is applicable to various types of automatic biochemical analyzers. Taking the Hitachi 7180 automatic biochemical analyzer as an example, the test conditions and operation steps are as follows:

[0043] Test conditions:

[0044] Temperature: 37℃ Dosage of Reagent R1 (μL) 270 Primary Wavelength (nm) 546 Dosage of Reagent R2 (μL) 90 Secondary Wavelength (nm) 700 Dosage of Sample (μL) 6 Reaction Direction Ascending Determination Method Two-Point Endpoint Method

[0045] Operation steps:

[0046]

[0047] Perform antibacterial experiments, anti-chylomicron interference experiments, and linearity experiments on the products in the above Examples 1-3, and compare them with commercially available creatinine detection kits in China.

[0048] (1) Antibacterial experiment:

[0049] Reflected by the microbial counting method, that is, after taking reagent R1 (labeled as R1 in the figure) and reagent R2 (labeled as R2 in the figure), they are respectively applied on the microbial culture medium and cultured overnight in an incubator at 37°C. The antibacterial situations of R1 and R2 are observed through the number of colonies, as Figure 1 shown.

[0050] From Figure 1 it can be seen that the commercially available creatinine kit R1 and reagent R2 have poor antibacterial ability, while Examples 1-3 have good antibacterial ability.

[0051] (2) Anti-chylomicron interference experiment:

[0052] Perform on-machine tests on different concentrations of chylomicron samples collected, and verify the anti-chylomicron interference ability of Examples 1-3 and commercially available creatinine detection kits by calculating the relative deviation and dilution ratio values. The specific results are shown in Tables 1 and 2.

[0053] Table 1 Measured values of chylomicron samples

[0054]

[0055] Table 2 Dilution ratio values of chylomicron samples (partial data)

[0056]

[0057]

[0058] From Table 1, it can be seen that the commercially available creatinine kit has poor anti-chylomicron sample interference ability. When the chylomicron concentration > 250 mg / dL, the deviation exceeds 10%. Examples 1-3 have better anti-chylomicron sample interference ability. When the chylomicron concentration ≤ 1000 mg / dL, it will not interfere with the measured value; for chylomicron samples > 500 mg / dL as shown in Table 2, the accuracy of the measured value can also be verified by 2-fold or 4-fold dilution. The relative deviation between the measured value after dilution and the original solution measured value is within ±10%, indicating that the detection results are reliable.

[0059] (3) Linear experiment:

[0060] Take creatinine standard products with concentrations of 0 (pure water), 1000, 3000, 5000, 7000, and 9000 μmol / L respectively, and use commercially available creatinine test kits and the creatinine test kits of Examples 1 - 3 to measure the creatinine concentration. The results are shown in Table 3:

[0061] Table 3 Comparison of linear ranges between commercially available creatinine test kits and the creatinine test kits of Examples 1 - 3

[0062] Theoretical Concentration (μmol / L) 0 1000 3000 5000 7000 9000 Commercially Available 0.0 998.5 2895.6 4786.3 6682.3 6931.4 Example 1 0.0 1011.3 2998.7 4956.8 6997.5 8997.6 Example 2 0.0 1009.2 3001.8 4989.3 7002.9 9008.2 Example 3 0.0 1002.4 3008.4 5003.7 7005.1 9010.4

[0063] As can be seen from Table 3, the linear high value of the commercially available creatinine test kit can only measure up to a concentration of 7000 μmol / L, while the linear high value of the reagents in Examples 1 - 3 can measure up to a concentration of 9000 μmol / L, indicating that Examples 1 - 3 have increased the linear range of the test kit. Conduct a linear correlation comparison for the reagents in Examples 1 - 3 at a concentration range of 0 - 9000 μmol / L, and it is found that their correlation is also good, as Figure 2 shown; the linear correlation is usually measured by the correlation coefficient R 2 ; the closer R 2 is to 1, the stronger the linear relationship between the two, and the better the correlation; the linear experiment of the test kit usually requires R 2 > 0.9900.

[0064] (4) Stability experiment

[0065] Open - package stability experiment: After opening the commercially available and the creatinine test kits of Examples 1 - 3 respectively, place them in the reagent storage compartment of an automatic biochemical analyzer, and monitor the measured values of the samples at 0 day, 7 days, 14 days, 21 days, and 30 days after opening, and calculate the relative deviation. The results are shown in Table 4.

[0066] Table 4 Results of relative deviation of stability after 30 - day opening of commercially available and the creatinine test kits of Examples 1 - 3

[0067]

[0068]

[0069] 37°C accelerated stability experiment: Put the commercially available and the creatinine test kits of Examples 1 - 3 into a 37°C water bath respectively, and detect the measured values of the samples at 0 day, 7 days, and 14 days in the 37°C water bath, and calculate the relative deviation. The results are shown in Table 5.

[0070] Table 5 Results of relative deviation of 37°C accelerated stability of commercially available and the creatinine test kits of Examples 1 - 3

[0071]

[0072]

[0073] Real-time stability experiment: For the commercially available and creatinine test kits of Examples 1-3, record the relevant reagent indicators after 0 months, 3 months, 6 months, and 12 months in real time, and calculate the relative deviation. The results are shown in Table 6.

[0074] Table 6 Relative deviation results of real-time 12-month stability of commercially available and creatinine test kits of Examples 1-3

[0075]

[0076]

[0077] From the above data, it can be seen that the stability of the creatinine test kits of Examples 1-3 is significantly better than that of the commercially available creatinine test kits, and it has good stability.

[0078] The above shows and describes the basic principles, main features and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic features of the present invention, the present invention can be implemented in other specific forms. Therefore, in any aspect, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claims involved.

[0079] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A creatinine detection kit with good stability and strong anti-interference ability, characterized in that, It includes the following components: reagent R1 and reagent R2; among them, Reagent R1 includes buffer 10 - 20 g / L, bacteriostatic agent 0.1 - 0.5 g / L, surfactant 0.2 - 0.5 g / L, enzyme activator 1 - 10 g / L, chromogen 0.5 - 1.5 g / L, creatinase 50 - 150 KU / L, sarcosine oxidase 10 - 20 KU / L and peroxidase 1 - 10 KU / L; Reagent R2 includes buffer 10 - 20 g / L, preservative 1 - 5 g / L, chromogenic reagent 0.5 - 1.5 g / L and creatinine enzyme 150 - 400 KU / L.

2. The creatinine detection kit with good stability and strong anti-interference according to claim 1, characterized in that, The bacteriostatic agent in the reagent R1 is gentamicin.

3. A creatinine detection kit with good stability and strong anti-interference according to claim 1, characterized in that, The surfactant of the reagent R1 is one or both of AEO - 7 and AEO - 9.

4. A creatinine detection kit with good stability and strong anti-interference according to claim 1, characterized in that, The enzyme activator of the reagent R1 is one or both of KCl and NaCl.

5. A creatinine detection kit with good stability and strong anti-interference, as described in claim 1, characterized in that The chromogen of the reagent R1 is sodium N - ethyl - N - (2 - hydroxy - 3 - sulfopropyl) - 3 - methylaniline.

6. A creatinine detection kit with good stability and strong anti-interference according to claim 1, characterized in that, The preservative of the reagent R2 is NaN3.

7. A creatinine detection kit with good stability and strong anti-interference according to claim 1, characterized in that, The chromogenic reagent of the reagent R2 is 4 - aminoantipyrine.

8. A creatinine detection kit with good stability and strong anti-interference according to claim 1, characterized in that, The buffer of the reagent R1 and reagent R2 is MOPSO buffer, and the pH value range is 6.5 - 8.

5.

9. A creatinine detection kit with good stability and strong anti-interference, as described in claim 1, wherein The volume ratio of the reagent R1 to the reagent R2 is 3:1.

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