The invention relates to 5apos; nucleotide enzyme calibrator and preparation method thereof

Abstract

The invention relates to the technical field of in-vitro detection reagents, in particular to a 5 '-nucleotidase calibrator and a preparation method thereof.The calibrator comprises 5'-nucleotidase, a borate buffering agent and animal serum, and the animal serum is selected from at least one of newborn calf serum, fetal calf serum, calf serum or sheep serum. The calibrator disclosed by the invention shows good stability and interchangeability, and ensures the detection accuracy of 5 '-nucleotidase.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of in vitro detection reagent, and particularly relates to a 5'-nucleotidase calibrator, a preparation method and application thereof. BACKGROUND

[0002] 5'-nucleotidase, also known as 5'-ribonucleotide phosphohydrolase (5'-NU or 5'-NT), is widely distributed in organs and tissues such as liver, gallbladder, pancreas, intestine, heart, brain, lung, kidney, pituitary, thyroid, prostate and testis, and also exists on the cell membrane. 5'-NT only exists in a small amount in normal serum, and the 5'-NT released into the blood circulation only comes from the liver and gallbladder tissue, because 5'-NT needs to be treated by high-concentration bile acid in the liver and gallbladder system before it can be released into the blood. Therefore, the change of the level of 5'-NT in serum is rarely affected by other diseases outside the liver, and the increase of the level of 5'-NT is often seen in liver and gallbladder diseases, such as hepatitis, cirrhosis, liver cancer and the like. The determination of serum 5'-NT in clinic provides an important reference for the diagnosis of liver and gallbladder diseases.

[0003] At present, biochemical reaction method is generally used to detect the level of serum 5'-NT in clinic, which mainly utilizes the specific catalytic effect of 5'-NT on 5'-nucleoside phosphate, detects the generation amount of reaction product or the rate of enzyme reaction by optical method (colorimetric method, ultraviolet spectrophotometry), and then compares with the standard curve measured by using different concentrations of calibrators to calculate the enzyme activity. The 5'-nucleotidase calibrator used in biochemical detection method is mostly in lyophilized form, but because 5'-NT in serum is prone to decay, it is difficult to be stored for a long time, resulting in that the existing calibrator has a short stable time after reconstitution. The development of long-acting and stable 5'-NT calibrator will significantly improve the accuracy and practicability of clinical detection, and is beneficial to the quality control of detection system and clinical popularization. SUMMARY

[0004] To solve the above technical problems, the present application realizes the effect of improving the storage stability and lyophilized reconstitution stability of 5'-nucleotidase calibrator by using borate buffer and animal serum as protective agents.

[0005] The first aspect of the present application provides a 5'-nucleotidase calibrator. The technical scheme is as follows: A 5'-nucleotidase calibrator, which is characterized in that it comprises 5'-nucleotidase, borate buffer and animal serum, and the animal serum is at least one selected from the group consisting of newborn bovine serum, fetal bovine serum, calf serum and sheep serum.

[0006] The calibrator as described above, wherein the borate buffer is selected from one of anhydrous sodium tetraborate, sodium tetraborate pentahydrate and sodium tetraborate decahydrate.

[0007] The calibrator as described above has a solution pH of 8.0-9.0.

[0008] The calibrator as described above has a solution pH of 8.7-8.9.

[0009] The calibrator as described above further comprises a plasticizing agent or / and a preservative.

[0010] The calibrator as described above, wherein the plasticizing agent is selected from at least one of trehalose, sucrose or mannitol.

[0011] The calibrator as described above, wherein the preservative is sodium azide.

[0012] The calibrator as described above, wherein the concentration of the borate buffer is 10-300 mmol / L and the content of the animal serum is 100-500 g / L.

[0013] The calibrator as described above, wherein the content of the 5'-nucleotidase is 30-100 U / L.

[0014] The calibrator as described above, wherein the content of the plasticizing agent is 10-50 g / L and the content of the preservative is 0.5-2.0 g / L.

[0015] The calibrator as described in any one of the above, which is a lyophilized dosage form, wherein the concentration of each component is the same as that in the original working solution before lyophilization or in the reconstituted solution of the lyophilized agent; preferably, the calibrator is further provided with a separately packaged purified water. The reconstituted solution is a working solution as specified in the product technical index, i.e. each bottle of lyophilized agent standard is reconstituted to a specified volume, and the working solution has the same concentration as the original working solution. Generally, the lyophilized agent is mixed and dissolved with purified water to obtain the reconstituted solution.

[0016] The second aspect of the present application provides a method for preparing a 5'-nucleotidase calibrator. The technical solution is as follows: The method for preparing a 5'-nucleotidase calibrator, which comprises lyophilizing an aqueous solution containing 5'-nucleotidase, a borate buffer and animal serum.

[0017] The third aspect of the present application provides a kit. The technical solution is as follows: The kit comprises a 5'-nucleotidase calibrator as described above.

[0018] The third aspect of the present application provides the use of the calibrator described above. The technical solution is as follows: The use of the 5'-nucleotidase calibrator as described above in the preparation of a detection method for 5'-nucleotidase in human serum samples for non-diagnostic purposes.

[0019] The use as described above, wherein the method for detecting human serum samples is a biochemical reaction detection method.

[0020] The fourth aspect of the present application provides a use of a protective agent. The technical solution is: The use of a protective agent composed of a borate buffer and an animal serum selected from at least one of neonatal bovine serum, fetal bovine serum, calf serum or sheep serum in the preparation of a 5'-nucleotidase calibrator. DETAILED DESCRIPTION

[0021] To make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0022] In this document, "and / or" includes any and all combinations of one or more of the associated items.

[0023] Abbreviations are as follows: HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Tris: Tris(hydroxymethyl)aminomethane 5'-NT: 5'-nucleotidase

[0024] In the present application, all the reagents are commercially available unless otherwise specified.

[0025] (I) Calibrator and preparation thereof A 5'-nucleotidase calibrator comprises 5'-nucleotidase, a borate buffer and an animal serum, wherein the animal serum is selected from at least one of neonatal bovine serum, fetal bovine serum, calf serum or sheep serum. The borate buffer is selected from one of anhydrous sodium tetraborate, sodium tetraborate pentahydrate and sodium tetraborate decahydrate. In addition, it further comprises a shaping agent and a preservative. The specific composition is as follows: Buffer: anhydrous sodium tetraborate, sodium tetraborate pentahydrate or sodium tetraborate decahydrate (borax), 10-300 mmol / L; Stabilizer: neonatal bovine serum, fetal bovine serum, calf serum or sheep serum, 100-500 g / L; Shaping agent: trehalose, sucrose or mannitol, 10-50 g / L; Enzyme: 5'-nucleotidase, 30-100 U / L; Preservative: sodium azide, 0.5-2.0 g / L; The solvent is purified water, and the pH of the solution is 8.0-9.0.

[0026] The preparation method of the calibrator is to dissolve the above components in a proportion to obtain a calibrator solution. Further, the calibrator solution is freeze-dried to obtain a freeze-dried agent.

[0027] (ii) Effect of calibrator composition on its performance With specific examples, the effect of calibrator composition on its performance was studied.

[0028] Different calibrator compositions Example 1 Lyophilized calibrator A 5'-nucleotidase calibrator was prepared with the original solution composition as shown in Table 1. The solution was stored after preparation with the amount of each component, or lyophilized to obtain the calibrator, and then reconstituted with purified water to obtain the standard working solution for testing.

[0029] Comparative Examples 1-4 The calibrator composition of Comparative Examples 1-4 was basically the same as that of Example 1, except that other substances were used to replace the borate buffer or / and no newborn calf serum was added. The specific composition is shown in Table 1. The missing components in the comparative examples were replaced with purified water, and the target pH of the solution was adjusted by the buffer in combination with acid or base. The newborn calf serum and 5'-nucleotidase were purchased from Sigma-Aldirch and Zhejiang Tianhang Biological Company, respectively.

[0030] Table 1 Composition of 5'-nucleotidase calibrator in Example 1 and Comparative Examples 1-4

[0031] Comparative Examples 5-9 The calibrator composition of Comparative Examples 5-9 was basically the same as that of Example 1, except that other substances were used to replace the protective agent component or / and no newborn calf serum was added. The specific composition is shown in Table 2. In Comparative Example 9, the content of trehalose was increased to replace the newborn calf serum. Compared with Example 1, the missing components in each comparative example were replaced with purified water, and the target pH of the solution was adjusted by the buffer in combination with sodium carbonate or sodium hydroxide.

[0032] Table 2 Composition of 5'-nucleotidase calibrator in Example 1 and Comparative Examples 5-9

[0033] Stability verification The fresh calibrator solution prepared at 4°C in each group was detected for the content of 5'-NT immediately after preparation, and after storage at 37°C for different time periods. The result measured immediately after preparation was taken as the reference, and the accelerated thermal stability of each group was studied.

[0034] At the same time, for different groups of calibrators, the lyophilized agent was reconstituted, and the reconstituted solution was stored at room temperature (25°C) for 1 day and 2 days, and then compared with the fresh reconstituted solution for testing to study the reconstitution stability.

[0035] The detection was performed using a 5'-NT kit (120 ml / kit, Zhongyuanhuiji Biotechnology Co., Ltd.) on a full-automatic biochemical analyzer (EXC2000, Zhongyuanhuiji Biotechnology Co., Ltd.). The test was performed according to the manufacturer's instructions, the main wavelength was selected as 546 nm, the reagent ratio was sample: R1: R2 = 6: 270: 90, and the average change rate of absorbance per minute within 3 min was determined.

[0036] The test results of the fresh calibrator solution at 4°C are given as the average of two measurements, and the test results of the calibrator stored at 37°C are presented as the deviation of the average of the measurements from the fresh calibrator solution, with a negative deviation indicating a lower measured value, as shown in Table 3. The calibrator of Example 1 was stored at 37°C for 11 days, and the measured value decreased by no more than 4%, while the decrease in each control was relatively large. Comparing Control Examples 1-4, it can be seen that the borate buffer (Control Example 4) has a lower decrease in the measured value of the calibrator than other buffer groups, and the decrease is slower, but the measured value decreases by more than 10% after 7 days of storage. Comparing Control Examples 5-9, it can be seen that the newborn calf serum group (Control Example 5) has a slightly lower decrease in the measured value of the calibrator than other sugar stabilizer groups, and the decrease is slower, but the measured value has decreased by more than 10% after 11 days of storage. The comprehensive comparison shows that the combination of borate buffer and newborn calf serum improves the accelerated thermal stability and maintains the enzyme activity of 5'-NT in the calibrator solution.

[0037] Table 3 Comparison of accelerated thermal stability test results of solutions of Example 1 and Control Examples 1-9

[0038] The test results of the calibrator lyophilized agent reconstituted solution are given as the average of two measurements, and the test results after storage of the reconstituted solution are presented as the deviation of the average of the measurements from the fresh calibrator solution, with a negative deviation indicating a lower measured value, as shown in Table 4. The initial measured values of the reconstituted solutions of the control groups have little deviation from Example 1, but the measured values after 1 day and 2 days of storage have decreased significantly to varying degrees, among which the decrease in Control Example 4 and Control Example 5 is relatively slow. After reconstitution of the lyophilized agent of Example 1, the measured value does not decrease significantly within 2 days, indicating that the combination of borate buffer and newborn calf serum improves the stability of the calibrator reconstituted solution.

[0039] Table 4 Comparison of reconstitution stability test results of solutions of Example 1 and Control Examples 1-9

[0040] By comparing different buffer and protective agent groups, it is found that sodium borate and newborn bovine serum have the most obvious effect on accelerating the thermal stability and improving the reconstitution stability of 5'-NT enzyme. The inventors found in the early stage that 5'-nucleotidase is prone to form multimers in solution, resulting in rapid decrease of absorbance and short stabilization period after reconstitution. The possible reasons for the protective agent system of the present application to improve the stability of 5'-NT enzyme are as follows: borate forms a reversible borate ester complex with the hydroxyl groups (such as serine and threonine residues) in the enzyme molecule, thereby locking the active conformation of the enzyme, preventing its aggregation and inactivation. At the same time, the high sodium ion environment of borate can weaken the hydrophobic interaction between 5'-NT enzyme molecules, reduce the aggregation and precipitation of enzyme molecules, "lock" the three-dimensional active conformation of the enzyme, avoid conformational unfolding caused by temperature and solvent changes, protect the spatial structure integrity of the active center, and prolong the half-life of the enzyme. In addition, borate or borate as a buffer substance can adjust the pH buffer environment of the reaction system, avoiding denaturation and inactivation of the enzyme under extreme pH conditions. The main mechanisms of the stability of newborn bovine serum to the enzyme are as follows: first, the carrier proteins such as albumin in the serum bind to the enzyme through hydrophobic interaction and hydrogen bond, reducing the aggregation and degradation of the enzyme molecules; second, the serum contains antioxidant substances such as glutathione, vitamin E, superoxide dismutase (SOD), which can scavenge free radicals in the system, reduce the damage of oxidative stress to the peptide chain of 5'-NT enzyme, and the protease inhibitors (such as α1 -antitrypsin) in the serum can inhibit the activity of trace amounts of proteases in the system, avoiding the hydrolytic degradation of 5'-NT enzyme, thereby maintaining the active state of the enzyme. These mechanisms work together to form a protective system of sodium borate and newborn bovine serum in the calibrator, which significantly improves the stability of the enzyme and maintains its catalytic efficiency. The inventors found that other borate buffer agents combined with fetal bovine serum, calf serum or sheep serum can also protect 5'-NT enzyme.

[0041] Further, the inter-bottle consistency of the calibrator of the present application and the adaptability to different batches of detection reagents are verified by interchangeability test. Five bottles of reconstitution solution of the calibrator of Example 1 are randomly taken as samples, and the 5'-NT enzyme level is detected according to the above method, and the test results are shown in Table 5. The inter-bottle consistency is excellent.

[0042] Table 5 Test results of inter-bottle consistency of calibrator

[0043] Using three batches of detection reagents to match the calibration sample reconstitution solution of Example 1, 20 random samples to be tested were tested to verify their adaptability, and the test results are shown in Table 6. As can be seen, the same sample is detected using different detection reagents-calibration samples, and the measured value deviation is small, and the calibration sample has high adaptability with different batches of detection reagents. The test results meet the technical requirements of the detection reagent: the sample enzyme content is between 5U / L-20U / L, and the absolute deviation is ≤3U / L; the sample enzyme content is between 20U / L-200U / L, and the relative deviation is ≤10%.

[0044] Table 6 Test results of calibration sample reconstitution solution and three different batches of detection reagents on samples

[0045] In summary, through the accelerated thermal stability, reconstitution stability, uniformity and three batches of reagent versatility verification experiments, it is proved that the combination of borate buffer system and newborn calf serum is used for 5'-NT calibration sample, and the calibration sample presents good stability and interchangeability, which ensures the accuracy of 5'-nucleotidase detection and meets the commercial value.

[0046] All data, reagents and steps in this document should be understood as illustrative rather than limiting, and although the present application has been described in connection with the above specific embodiments, many modifications and other variations will be apparent to those skilled in the art. All such modifications and other variations also fall within the scope and spirit of the present application.

Claims

1. A 5'-nucleotidase calibrator, characterized in that, The animal serum is at least one selected from the group consisting of newborn calf serum, fetal calf serum, calf serum, and sheep serum.

2. The calibrant of claim 1, wherein, The borate buffer is at least one selected from the group consisting of anhydrous sodium tetraborate, sodium tetraborate pentahydrate, and sodium tetraborate decahydrate.

3. The calibrator according to claim 1 or 2, wherein the pH of the solution is 8.0-9.0, preferably 8.7-8.

9.

4. The calibrator according to claim 1 or 2, further comprising a plasticizing agent or / and a preservative.

5. The calibrator according to claim 4, wherein the plasticizing agent is at least one selected from the group consisting of trehalose, sucrose, and mannitol; and / or the preservative is sodium azide.

6. The calibrator according to claim 1 or 2, wherein the concentration of the borate buffer is 10-300 mmol / L, and the content of the animal serum is 100-500 g / L; and / or the content of the 5'-nucleotidase is 30-100 U / L.

7. The calibrator according to claim 6, which is in a lyophilized form, wherein the concentration of each component is the concentration in the original solution before lyophilization or in the reconstituted solution.

8. A method for preparing a 5'-nucleotidase calibrator, comprising the steps of: a) preparing an aqueous solution comprising 5'-nucleotidase, a borate buffer, and an animal serum; and b) lyophilizing the aqueous solution.

9. A kit comprising the 5'-nucleotidase calibrator according to any one of claims 1-7.

8. A method for the preparation of a calibrant according to claim 7, characterized in that, 10. Use of a protective agent comprising a borate buffer and an animal serum in the preparation of a 5'-nucleotidase calibrator, wherein the animal serum is at least one selected from the group consisting of newborn calf serum, fetal calf serum, calf serum, and sheep serum. ​ ​

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