Alkaline phosphatase detection reagent and application thereof

Abstract

The invention provides an alkaline phosphatase detection reagent based on a BCIP / NBT method. The alkaline phosphatase detection reagent comprises a reagent R1 and a reagent R2, wherein the reagent R1 is prepared from gelatin and 5-bromo-4-chloro-3-indolyl phosphate, and the reagent R2 is prepared from a reagent R3 and a reagent R4; and the reagent R2 contains nitrotetrazole blue chloride. The gelatin is added into the reagent R1, so that the thermal stability of the reagent R1 is enhanced, and the problems that the current alkaline phosphatase detection kit based on a BCIP / NBT method is short in validity period and difficult to transport and store are solved. In addition, the detection reaction system is mild, the reagent does not involve strong acid or strong alkali, and the possibility that the reagent hurts producers and users is avoided. Meanwhile, the detection reagent is simple in composition, only the reagent R1 and the reagent R2 are used, a full-automatic biochemical analyzer can be used for detecting the content of alkaline phosphatase, operation is simpler, large-scale detection can be met, and the result is accurate.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and more specifically, relates to an alkaline phosphatase detection reagent and application thereof. Background Art

[0002] Alkaline phosphatase (ALP or AKP) is an enzyme widely distributed in human liver, bones, intestines, kidneys and placenta. Alkaline phosphatase is an enzyme that can dephosphorylate the corresponding substrate, that is, it removes the phosphate group on the substrate molecule by hydrolyzing the phosphate monoester and generates phosphate ions and free hydroxyl groups. Such substrates include nucleic acids, proteins, alkaloids, etc. It is not a single enzyme, but a group of isozymes. Six isozymes, AKP1, AKP2, AKP3, AKP4, AKP5 and AKP6, have been found, and they are derived from different tissues and organs. Alkaline phosphatase plays an important physiological function in the human body, mainly including: participating in bone metabolism and the synthesis of extracellular matrix, such as collagen and elastin; playing an important role in bone growth and repair; helping to maintain physiological processes such as human lipid metabolism and nerve conduction. Therefore, it is of great significance to study the detection reagent of alkaline phosphatase. In addition, since the detection of alkaline phosphatase is a common clinical test item with a large demand for detection, there is an urgent need to develop an alkaline phosphatase detection kit suitable for high-throughput detection by fully automatic biochemical analyzers.

[0003] At present, the detection methods of alkaline phosphatase mainly include continuous detection method (NPP substrate-AMP buffer method), BCIP / NBT method, etc. Among them, the BCIP / NBT method has relatively simple components and is suitable for high-throughput detection of alkaline phosphatase content, so it has better application potential. The basic principle of the BCIP / NBT method is that under the catalysis of alkaline phosphatase, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) will be hydrolyzed to produce a highly reactive product, which will react with nitro blue tetrazolium chloride (NBT) to form an insoluble dark blue to blue-purple NBT-formazan, thereby developing color and realizing the detection of alkaline phosphatase.

[0004] However, the current test kits based on the BCIP / NBT principle have problems such as poor thermal stability and short shelf life, and also have relatively high requirements for transportation conditions. Summary of the invention

[0005] The present invention aims to solve the problem of poor thermal stability of the BCIP / NBT method detection system in the above-mentioned prior art, and provide an alkaline phosphatase detection reagent combination with excellent thermal stability.

[0006] The purpose of the present invention is to provide an alkaline phosphatase detection reagent based on the BCIP / NBT method and application thereof.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] The invention provides an alkaline phosphatase detection reagent based on a BCIP / NBT method, comprising a reagent R1 and a reagent R2; wherein the reagent R1 contains gelatin and 5-bromo-4-chloro-3-indolyl phosphate; and the reagent R2 contains nitro blue tetrazolium chloride.

[0009] The present invention has concluded through exploration and research that adding gelatin to the alkaline phosphatase detection reagent of the BCIP / NBT method can significantly improve the stability of the reagent and has very good application value.

[0010] As a further optional embodiment, the concentration of the 5-bromo-4-chloro-3-indolyl phosphate in reagent R1 is 0.1-10 mM, the concentration of the gelatin in reagent R1 is 0.1-10 g / L, and the concentration of the nitro blue tetrazolium chloride in reagent R2 is 1-20 mM.

[0011] As an optional embodiment, the solvent of the reagent R1 and the reagent R2 is water.

[0012] In the embodiment of the present invention, the concentration of the 5-bromo-4-chloro-3-indolyl phosphate in reagent R1 is 0.74 mM, the concentration of the gelatin in reagent R1 is 0.25-2 g / L (preferably 1-2 g / L), and the concentration of the nitro blue tetrazolium chloride in reagent R2 is 10 mM.

[0013] As an optional embodiment, the pH value of the reagent R1 is 8.5-10.0, and the pH value of the reagent R2 is 5.5-7.0.

[0014] As an alternative embodiment, the pH value of the reagent R1 is 9.0, and the pH value of the reagent R2 is 6.5.

[0015] As an optional embodiment, the reagent R1 further comprises a buffer and an inorganic salt; and the reagent R2 further comprises citric acid, ascorbic acid oxidase and a surfactant.

[0016] As an optional embodiment, the reagent R1 and the reagent R2 further include a preservative.

[0017] As an optional embodiment, the inorganic salt is at least one of sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, zinc sulfate, magnesium acetate, and zinc acetate.

[0018] As an optional embodiment, the buffer is at least one of borate, citrate, phosphate, tris(hydroxymethylaminomethane), piperazine-1,4-diethanesulfonic acid, and 3-morpholinepropanesulfonic acid.

[0019] As an optional embodiment, the surfactant is at least one of Triton TX 100, Tween 20, Triton X 405, and Bridger 35.

[0020] As an optional embodiment, the preservative is at least one of thimerosal, sodium benzoate, gentamicin, and Proclin300.

[0021] As an optional embodiment, the concentration of the buffer in reagent R1 is 50-500mM, the concentration of the inorganic salt in reagent R1 is 0.01-10mM, the concentration of citric acid in reagent R2 is 5-500mM, the concentration of ascorbic acid oxidase in reagent R2 is 1-100U / L, the concentration of the surfactant in reagent R2 is 0.1-2mL / L, and the concentration of the preservative in both reagent R1 and reagent R2 is 0.1-2mL / L.

[0022] In the embodiment of the present invention, the buffer is tris(hydroxymethyl)aminomethane, and its concentration in reagent R1 is 100 mM; the inorganic salts are anhydrous magnesium sulfate, zinc sulfate heptahydrate, and sodium chloride, and their concentrations in reagent R1 are 0.5 mM, 0.03 mM, and 10 mM, respectively; the concentration of citric acid in reagent R2 is 50 mM; the concentration of ascorbic acid oxidase in reagent R2 is 10 U / L; the surfactant is Triton TX 100, and its concentration in reagent R2 is 0.5 mL / L; the preservative is Proclin 300, and its concentrations in both reagent R1 and reagent R2 are 0.5 mL / L.

[0023] The use of the above reagents in the preparation of an alkaline phosphatase detection kit should also be within the protection scope of the present invention.

[0024] As an optional implementation scheme, the preparation method of reagent R1 is: add materials into water in the order of buffer, inorganic salt, stabilizer, BCIP, and preservative, add the next material after the previous material is completely dissolved, and adjust the pH value after the materials are completely dissolved to prepare it.

[0025] As an optional implementation scheme, the preparation method of reagent R2 is: add citric acid, ascorbic acid oxidase, NBT, surfactant, and preservative into water in this order, add the next material after the previous material is completely dissolved, and adjust the pH value after the material is completely dissolved to prepare it.

[0026] The present invention has the following beneficial effects:

[0027] The present invention enhances the thermal stability of the reagent R1 by adding gelatin to the reagent R1, thereby overcoming the problems of the short validity period and difficult transportation and storage of the alkaline phosphatase detection kit based on the current BCIP / NBT method. In addition, the detection reaction system of the present invention is mild, and the reagent does not involve strong acids and strong bases, thereby avoiding the possibility of the reagent causing harm to the producer and the user. At the same time, the detection reagent of the present invention has a simple composition, and only has two reagents, reagent R1 and reagent R2. A fully automatic biochemical analyzer can be used to detect the alkaline phosphatase content, and the operation is simpler, and it can meet the needs of large-scale detection, and the results are accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure is a comparison of the results of alkaline phosphatase detection by the control kit and the kit of Example 1. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0030] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0031] The manufacturer of the alkaline phosphatase kit (NPP substrate-AMP buffer method) is Sichuan MacBiotech Co., Ltd., and the product number is CH0101203.

[0032] 5-Bromo-4-chloro-3-indolyl phosphate (BCIP), CAS No.: 217656-69-4, structural formula:

[0033]

[0034] Nitro blue tetrazolium chloride (NBT), CAS number: 298-83-9, structural formula:

[0035]

[0036] Tris(hydroxymethyl)aminomethane, CAS number: 77-86-1, structural formula:

[0037] Gelatin, CAS number: 9000-70-8.

[0038] Example 1 Alkaline phosphatase assay kit

[0039] First add some purified water to a beaker on a magnetic stirrer and stir at medium speed. Add materials in the order of components from top to bottom in Table 1. Add the next material after the previous material is completely dissolved. Stir until the material is completely dissolved. Adjust the pH value and make up to the final volume. Reagent R1 and reagent R2 are prepared separately.

[0040] Table 1 Components of reagent R1 and reagent R2 in the kit

[0041]

[0042]

[0043] Example 2 Alkaline phosphatase assay kit

[0044] First add some purified water to a beaker on a magnetic stirrer and stir at medium speed. Add materials in the order of components from top to bottom in Table 2. Add the next material after the previous material is completely dissolved. Stir until the material is completely dissolved. Adjust the pH value and make up to the final volume. Reagent R1 and reagent R2 are prepared separately.

[0045] Table 2 Components of reagent R1 and reagent R2 in the kit

[0046]

[0047] Example 3 Alkaline phosphatase assay kit

[0048] First, add some purified water to a beaker on a magnetic stirrer and stir at medium speed. Add materials in the order of components from top to bottom in Table 3. Add the next material after the previous material is completely dissolved. Stir until the material is completely dissolved. Adjust the pH value and make up to the final volume. Reagent R1 and reagent R2 are prepared separately.

[0049] Table 3 Components of reagent R1 and reagent R2 in the kit

[0050]

[0051] Example 4 Alkaline Phosphatase Assay Kit

[0052] First add some purified water to a beaker on a magnetic stirrer and stir at medium speed. Add materials in the order of components from top to bottom in Table 4. Add the next material after the previous material is completely dissolved. Stir until the material is completely dissolved. Adjust the pH value and make up to the final volume. Reagent R1 and reagent R2 are prepared separately.

[0053] Table 4 Components of reagent R1 and reagent R2 in the kit

[0054]

[0055] Example 5 Alkaline phosphatase assay kit

[0056] First add some purified water to a beaker on a magnetic stirrer and stir at medium speed. Add materials in the order of components from top to bottom in Table 5. Add the next material after the previous material is completely dissolved. Stir until the material is completely dissolved. Adjust the pH value and make up to the final volume. Reagent R1 and reagent R2 are prepared separately.

[0057] Table 5 Components of reagent R1 and reagent R2 in the kit

[0058]

[0059] Comparative Example 1 Alkaline Phosphatase Assay Kit

[0060] First add some purified water to a beaker on a magnetic stirrer and stir at medium speed. Add materials in the order of components from top to bottom in Table 6. Add the next material after the previous material is completely dissolved. Stir until the material is completely dissolved. Adjust the pH value and make up to the final volume. Reagent R1 and reagent R2 are prepared separately.

[0061] Table 6 Components of reagent R1 and reagent R2 in the kit

[0062]

[0063]

[0064] Comparative Example 2 Alkaline Phosphatase Assay Kit

[0065] First add some purified water to a beaker on a magnetic stirrer and stir at medium speed. Add materials in the order of components from top to bottom in Table 7. Add the next material after the previous material is completely dissolved. Stir until the material is completely dissolved. Adjust the pH value and make up to the final volume. Reagent R1 and reagent R2 are prepared separately.

[0066] Table 7 Components of reagent R1 and reagent R2 in the kit

[0067]

[0068] Comparative Example 3 Alkaline Phosphatase Assay Kit

[0069] First add some purified water to a beaker on a magnetic stirrer and stir at medium speed. Add materials in the order of components from top to bottom in Table 8. Add the next material after the previous material is completely dissolved. Stir until the material is completely dissolved. Adjust the pH value and make up to the final volume. Reagent R1 and reagent R2 are prepared separately.

[0070] Table 8 Components of reagent R1 and reagent R2 in the kit

[0071]

[0072]

[0073] Comparative Example 4 Alkaline Phosphatase Assay Kit

[0074] First add some purified water to a beaker on a magnetic stirrer and stir at medium speed. Add materials in the order of components from top to bottom in Table 9. Add the next material after the previous material is completely dissolved. Stir until the material is completely dissolved. Adjust the pH value and make up to the final volume. Reagent R1 and reagent R2 are prepared separately.

[0075] Table 9 Components of reagent R1 and reagent R2 in the kit

[0076]

[0077] Experimental Example 1: Alkaline Phosphatase Measurement Method

[0078] The test was performed using a fully automatic biochemical analyzer (Hitachi 3500), with detection wavelengths: main wavelength 340 nm, secondary wavelength 405 nm, and measurement mode: rate method.

[0079] The method for detecting alkaline phosphatase of the kit of the present invention is as follows: blank tubes, quality control tubes, calibration tubes and sample tubes are prepared according to Table 10, mixed after preparation, incubated at 37°C for 5 minutes, then 80 μL of reagent R2 is added and mixed, and absorbance A1 is measured after incubation at 37°C for 2 minutes; incubation is continued for 3 minutes, absorbance A2 of each tube is recorded, and ΔA / min is calculated.

[0080] Table 10 Reagent preparation of the kit

[0081] Blank tube Quality Control Calibration tube Sample tube R1(μL) 240 240 240 240 Purified water (μL) 12 - - - Quality control product (μL) - 12 - - Calibrator (μL) - - 12 Sample (μL) - - - 12

[0082] Calculation method of alkaline phosphatase in samples:

[0083]

[0084] Where:

[0085] ΔAsample / min——the rate of change of absorbance of the sample;

[0086] ΔA calibrator / min——the rate of change of absorbance of the calibrator;

[0087] Using the quality control serum samples as the test samples, the kits of Example 1 and Comparative Examples 1-4 were subjected to 37°C accelerated tests. The 37°C accelerated test is specifically as follows: Reagent R1 and reagent R2 are placed in a 37°C constant temperature drying oven for acceleration, and the alkaline phosphatase content of the reagent R1 and reagent R2 test samples with different accelerated days are taken. The alkaline phosphatase content measured by the reagents for different accelerated days is compared with the alkaline phosphatase content measured by the reagents on day 0 (reagent R1 and reagent R2 were not subjected to 37°C accelerated test). The relative deviation is within ±10%, which is considered to be a good stability of the kit. The number of heating days for the thermal stability test is 37°C heating for one day, which is equivalent to the product being stored at 2-8°C for 1.8 months.

[0088] The reagents R1 and R2 in the kits of Example 1 and Comparative Examples 1-4 are provided to a fully automatic biochemical analyzer, and the parameters of the analyzer are set according to the method for detecting alkaline phosphatase in the kit of the present invention, and then a 37°C accelerated test is performed. The test results are shown in Tables 11-15, and the results show that Example 1 can be stable for 10 days, Comparative Example 1 can be stable for 4 days, Comparative Example 2 can be stable for 3 days, Comparative Example 3 can be stable for 7 days, and Comparative Example 4 can be stable for 3 days. The kit of Example 1 can be stable for 10 days under the 37°C accelerated test, which is equivalent to a validity period of 18 months at 2-8°C, indicating that the kit of Example 1 has good stability and can meet long-term storage requirements.

[0089] Table 11 Results of alkaline phosphatase assay in samples using the kit of Example 1

[0090]

[0091] Table 12 Results of alkaline phosphatase determination in samples by the kit of comparative example 1

[0092]

[0093] Table 13 Comparative Example 2 Test kit results for alkaline phosphatase in samples

[0094]

[0095] Table 14 Results of alkaline phosphatase determination in samples by the kit of comparative example 3

[0096]

[0097] Table 15 Comparative Example 4 Test kit results for alkaline phosphatase in samples

[0098]

[0099]

[0100] Experimental Example 2

[0101] The kits of Examples 1-5 were respectively subjected to calibration precision tests, and the method for detecting alkaline phosphatase was referred to Experimental Example 1. (The lower the coefficient of variation, the smaller the volatility of the test results and the more stable the results).

[0102] The test results are shown in Table 16. The results show that with the change of gelatin concentration in reagent R1, the coefficient of variation of the precision of the two concentration level calibrants presents different trends. The coefficient of variation of the precision of Example 1, Example 4 and Example 5 is significantly smaller than that of Example 2 and Example 3, indicating that with the increase of gelatin concentration in the reagent, the detection precision of the kit for alkaline phosphatase is higher; among which the precision of Example 4 and Example 5 relative to Example 1 is consistent, indicating that the gelatin concentration in the reagent R1 of Example 1 is the optimal concentration in consideration of the comprehensive cost and other factors in order to meet the detection needs of the kit.

[0103] Table 16 Coefficient of variation of alkaline phosphatase detection kits in different embodiments

[0104]

[0105] Experimental Example 3

[0106] 40 fresh serum samples were taken as samples, and the alkaline phosphatase content was detected using the alkaline phosphatase kit (NPP substrate-AMP buffer method) as a control kit, and the detection method was carried out according to its instructions. At the same time, the alkaline phosphatase content of the same samples was detected using the alkaline phosphatase kit (NBT method) of Example 1.

[0107] The regression equation, correlation coefficient and relative deviation of the two test values ​​were calculated by linear regression method. The results are shown in Table 17 and Figure 1 As shown, the results show that the relative deviations of the two kits are within ±10%. Figure 1 The regression equation of the two groups of reagents is y = 1.0238x + 0.4101 and the correlation coefficient R 2 =0.9998, both the accuracy and correlation are very high.

[0108] Table 17 Relative deviation analysis results

[0109]

[0110]

[0111] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An alkaline phosphatase detection reagent based on the BCIP / NBT method, characterized in that: The invention comprises reagent R1 and reagent R2; wherein the reagent R1 contains gelatin and 5-bromo-4-chloro-3-indolyl phosphate; and the reagent R2 contains nitro tetrazolium blue chloride.

2. The reagent according to claim 1, characterized in that The concentration of the 5-bromo-4-chloro-3-indolyl phosphate in the reagent R1 is 0.1-10 mM, the concentration of the gelatin in the reagent R1 is 0.1-10 g / L, and the concentration of the nitro blue tetrazolium chloride in the reagent R2 is 1-20 mM.

3. The reagent according to claim 1 or 2, characterized in that The reagent R1 further comprises a buffer and an inorganic salt; the reagent R2 further comprises citric acid, ascorbic acid oxidase and a surfactant.

4. The reagent according to claim 3, characterized in that The reagents R1 and R2 also include a preservative.

5. The reagent according to claim 3, characterized in that The inorganic salt is at least one of sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, zinc sulfate, magnesium acetate, and zinc acetate.

6. The reagent according to claim 3, characterized in that The buffer is at least one of borate, citrate, phosphate, tris(hydroxymethyl)aminomethane, piperazine-1,4-diethanesulfonic acid, and 3-morpholinepropanesulfonic acid.

7. The reagent according to claim 3, characterized in that The surfactant is at least one of Triton TX 100, Tween 20, Triton X 405, and Bridger 35.

8. The reagent combination according to claim 4, characterized in that: The preservative is at least one of thimerosal, sodium benzoate, gentamicin and Proclin 300.

9. The reagent according to claim 4, characterized in that The concentration of the buffer in reagent R1 is 50-500mM, the concentration of the inorganic salt in reagent R1 is 0.01-10mM, the concentration of citric acid in reagent R2 is 5-500mM, the concentration of ascorbic acid oxidase in reagent R2 is 1-100U / L, the concentration of the surfactant in reagent R2 is 0.1-2mL / L, and the concentration of the preservative in both reagent R1 and reagent R2 is 0.1-2mL / L.

10. Use of the reagent according to any one of claims 1 to 9 in the preparation of an alkaline phosphatase detection kit.

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