NADH protective agent and preparation method thereof

Abstract

The present invention provides a NADH protective agent and a preparation method thereof, wherein the NADH protective agent comprises: a buffer, a reducing agent, ethylene glycol and BSA; wherein the pH value of the NADH protective agent is 9.0-10.0. In the present invention, the reducing agent is used to protect NADH. When an oxidation-reduction reaction occurs, the reducing agent participates in the reaction, while NADH remains stable and does not participate in the reaction. This method is used to achieve the purpose of protecting NADH. A protective agent component with strong reduction, good stability and low price is selected to solve the problems of high cost and poor stability of the protection system. By completely abandoning the idea of ​​enzyme protective agents, the problem of enzyme interference in actual use is avoided.

Description

Technical Field

[0001] The invention belongs to the field of biological preparations, and specifically relates to a NADH protective agent and a preparation method thereof. Background Art

[0002] The commonly used NADH protection system is the glucose-6-phosphate dehydrogenase system. The main components of this system are glucose, phosphate, G-6-PDH, and HK (hexokinase). The main principle is that NAD+ is the oxidized form of NADH. Glucose forms glucose-6-phosphate under the catalysis of hexokinase, and then glucose-6-phosphate forms phosphogluconolactone under the action of glucose-6-phosphate dehydrogenase, and NAD+ is reduced to NADH, thereby maintaining the stability of the relative concentration of NADH. However, since this system uses two enzymes, it has the following disadvantages in practical applications: 1. High cost: The market price of the two enzymes needed in this system is relatively high, which increases the cost in actual use; 2. Poor stability: Since the enzyme itself is an unstable substance, the enzyme itself will also decompose and denature during long-term storage, and eventually inactivate, thereby weakening or ineffective the protection ability of the protection system. 3. Interference reaction. Because the essence of the glucose-6-phosphate dehydrogenase system is to achieve the purpose of protection by reducing NAD+ to NADH. In reactions involving NADH, if the amount of enzyme in the protection system is inappropriate, it will interfere with the reaction of NADH to NAD+ and interfere with the final result. Summary of the invention

[0003] In view of this, the present invention provides a NADH protective agent and a preparation method thereof, aiming to provide a NADH protective agent with low preparation cost and good protective effect, thereby avoiding the problem of enzyme interference in actual use.

[0004] To achieve the above object, the present invention provides a NADH protective agent, which comprises: a buffer, a reducing agent, ethylene glycol and BSA;

[0005] Wherein, the pH value of the NADH protective agent is 9.0-10.0.

[0006] Optionally, the reducing agent includes at least one of glucose, mannitol, glycerol and potassium sorbate.

[0007] Optionally, the concentration of the reducing agent is 30 g / L~50 g / L.

[0008] Optionally, in the NADH protective agent, the concentration of ethylene glycol is 200 ml / L~400 ml / L.

[0009] Optionally, in the NADH protective agent, the concentration of BSA is 0.5 g / L~2 g / L.

[0010] Optionally, the buffer includes any one of Tris buffer, PBS buffer and Good's buffer.

[0011] Optionally, the concentration of the buffer is 50mmol / L~100mmol / L.

[0012] In addition, the present invention provides a method for preparing the above-mentioned NADH protective agent, and the method for preparing the NADH protective agent comprises the following steps:

[0013] Adding a buffer solution to deionized water to obtain a first solution;

[0014] adding a reducing agent, ethylene glycol and BSA to the first solution in sequence to obtain a second solution;

[0015] The pH value of the second solution is adjusted to 9.0-10 to obtain a NADH protective agent.

[0016] In the present invention, other reducing substances are used to protect NADH. When the redox reaction occurs, other reducing substances participate in the reaction, while NADH remains stable and does not participate in the reaction. This method is used to achieve the purpose of protecting NADH. A protective agent component with strong reduction, good stability and low price is selected to solve the problem of high cost and poor stability of the protection system. By completely abandoning the idea of ​​enzyme protective agent, the problem of enzyme interference in actual use is avoided. At the same time, high pH is used to stabilize NADH. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The present invention provides a flow chart of the preparation method of the NADH protective agent.

[0019] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0020] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0021] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0022] The commonly used protection system is the glucose-6-phosphate dehydrogenase system, but since this system uses two enzymes, in practical applications, because the glucose-6-phosphate dehydrogenase system essentially achieves the purpose of protection by reducing NAD+ to NADH, in the reaction involving NADH, if the amount of enzyme in the protection system is inappropriate, it will interfere with the reaction of NADH to NAD+, and interfere with the final result.

[0023] In view of this, the present invention provides a NADH protective agent. The following is an embodiment of the NADH protective agent provided by the present invention.

[0024] The NADH protective agent comprises the following components:

[0025] Buffer, reducing agent, ethylene glycol and BSA; wherein the pH value of the NADH protective agent is 9.0-10.0.

[0026] In the present invention, other reducing substances are used to protect NADH. When the redox reaction occurs, the reducing substances in the reducing agent participate in the reaction, while NADH remains stable and does not participate in the reaction. This method is used to achieve the purpose of protecting NADH. The reducing agent component with strong reduction, good stability and low price is selected to solve the problem of high cost and poor stability of the NADH protection system. By completely abandoning the idea of ​​enzyme protecting agents, the problem of enzyme interference in actual use is avoided. At the same time, high pH is used to stabilize NADH.

[0027] In some embodiments, the buffer includes any one of tris (hydroxymethylaminomethane) (Tris buffer), phosphate buffer (PBS buffer) and zwitterionic buffer (Good's buffer), and the use of the above buffer can further improve the stability of the pH environment of the solvent. Specifically, the concentration of any of the above buffers is 50mmol / L~100mmol / L.

[0028] In some embodiments, the reducing agent includes at least one of glucose, mannitol, glycerol and potassium sorbate. The use of the above reducing substances avoids oxidative damage to NADH and improves the stability of NADH storage.

[0029] In some embodiments, the concentration of ethylene glycol is 200 ml / L to 400 ml / L; the concentration of BSA is 0.5 g / L to 2 g / L. Ethylene glycol and BSA are used to protect the stability of NADH while maintaining the stability of the relative concentration of NADH.

[0030] In addition, the present invention also provides a method for preparing the above-mentioned NADH protective agent, and the method for preparing the NADH protective agent comprises:

[0031] S10, adding a buffer solution to deionized water to obtain a first solution;

[0032] S20, sequentially adding a reducing agent, ethylene glycol and BSA to the first solution to obtain a second solution;

[0033] S30, adjusting the pH value of the second solution to 9.0-10 to obtain a NADH protective agent.

[0034] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0035] Embodiment 1 to Embodiment 4

[0036] Examples 1 to 4 provide a NADH protective agent, respectively, and the specific components are shown in Table 1:

[0037] Table 1 Composition and final concentration of NADH protective agent in Examples 1 to 4

[0038]

[0039] Examples 1 to 4 also provide a method for preparing the above-mentioned NADH protective agent, and the specific operation method is:

[0040] S10, adding a buffer solution to deionized water to obtain a first solution;

[0041] S20, sequentially adding a reducing agent, ethylene glycol and BSA to the first solution to obtain a second solution;

[0042] S30, adjusting the pH value of the second solution to 9.0-10.0 to obtain a NADH protective agent.

[0043] Wherein, the preparation method is carried out according to the substances described in the above table.

[0044] Embodiment 5 to Embodiment 8

[0045] Examples 5 to 8 provide a NADH protective agent, respectively, and the specific components are shown in Table 2:

[0046] Table 2 Composition and final concentration of NADH protective agent in Examples 5 to 8

[0047]

[0048] Examples 5 to 8 also provide a method for preparing the above-mentioned NADH protective agent, and the specific operation method is:

[0049] S10, adding a buffer solution to deionized water to obtain a first solution;

[0050] S20, sequentially adding a reducing agent, ethylene glycol and BSA to the first solution to obtain a second solution;

[0051] S30, adjusting the pH value of the second solution to 9.0-10.0 to obtain a NADH protective agent.

[0052] Wherein, the preparation method is carried out according to the substances described in the above table.

[0053] Comparative Example 1

[0054] This comparative example provides an antibody storage solution having substantially the same components as those of Example 1, except that it does not contain a reducing agent.

[0055] Comparative Example 2

[0056] This comparative example provides an antibody storage solution having substantially the same components as those of Example 1, except that it does not contain ethylene glycol.

[0057] Comparative Example 3

[0058] This comparative example provides an antibody storage solution having substantially the same components as those of Example 1, except that it does not contain BSA.

[0059] Test Example 1

[0060] 1. Use a graduated pipette to take blood type Lewis A antibody and add it to the NADH protection solution of Examples 1 to 8 and Comparative Example 1 respectively, and make the concentration of blood type Lewis A antibody 500 μg / mL. One part is stored at 4° C., and the other part is tested for titer. After testing different antibody protection solutions, the initial titer is 1:512.

[0061] 2. One month later, the portion stored at 4°C was taken out and the titer was tested again according to the above steps. The ratio of the latter titer to the previous titer was calculated, which was the activity retention rate. At the same time, the portion not subjected to the experiment was preserved at 4°C again, repeatedly frozen and thawed during the period, and taken out again after two months for a titer test. The ratio of the detected titer to the initial titer was the activity retention rate. The test results are shown in Table 3.

[0062] Table 3 Results of titer test

[0063]

[0064] As can be seen from Table 3, compared with Comparative Example 1, Examples 1 to 8 used a reducing agent as a protective agent, and compared with Comparative Example 2, an antioxidant was added, so that the activity retention rate of the antibody was 100% after being stored at 4°C for one month, and the activity retention rate after repeated freezing and thawing was above 50%. At the same time, when the pH value of DADH was 9 and the protective agent contained glucose, mannitol, glycerol and potassium sorbate, the activity retention rate of the antibody was 100% after repeated freezing and thawing.

[0065] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A NADH protective agent, It is characterized in that The NADH protective agent comprises a buffer, a reducing agent, ethylene glycol and BSA; wherein the pH value of the NADH protective agent is 9.0-10.0; The reducing agent comprises at least one of glucose, mannitol, glycerol and potassium sorbate; The concentration of the reducing agent is 30 g / L to 50 g / L; In the NADH protective agent, the concentration of ethylene glycol is 200ml / L to 400ml / L; In the NADH protective agent, the concentration of BSA is 0.5 g / L to 2 g / L; The buffer comprises any one of Tris buffer, PBS buffer and Good's buffer; The concentration of the buffer solution is 50 mmol / L to 100 mmol / L; The NADH protective agent is prepared by the following method, comprising the following steps: Adding a buffer solution to deionized water to obtain a first solution; adding a reducing agent, ethylene glycol and BSA to the first solution in sequence to obtain a second solution; The pH value of the second solution is adjusted to 9.0-10 to obtain a NADH protective agent.

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