阅读说明：本技术 一种脑特异性蛋白产物9.5的冻干方法 (Freeze-drying method of brain specific protein product 9.5 ) 是由 李博飞 刘聪 杨涛 张广俊 冯玉静 孙佳 郑兴华 张明琛 于 2021-09-27 设计创作，主要内容包括：本发明属于脑特异性蛋白产物9.5保存技术领域,具体涉及一种脑特异性蛋白产物9.5的冻干方法。所述冻干方法包括以下步骤：取脑特异性蛋白产物9.5和缓冲液配制成溶液,设置冻干程序进行冻干,所述缓冲液的组成包括以下组分：4-吗啉乙磺酸、NaCl、ZnCl-(2)、MgCl-(2)·6H-(2)o、牛血清白蛋白、酪蛋白、甘氨酸、聚乙二醇、海藻糖。能够有效保存脑特异性蛋白产物9.5,冻干品复融后可有效保存其活性,提高检测灵敏度；所述冻干保护方法对脑特异性蛋白产物9.5的保存具有较好的保存效果,可以使得PGP9.5在4-8℃储存期达到18个月、在-20℃储存期达到3年。(The invention belongs to the technical field of preservation of a brain specific protein product 9.5, and particularly relates to a freeze-drying method of the brain specific protein product 9.5. The lyophilization process comprises the steps of: preparing a brain specific protein product 9.5 and a buffer solution into a solution, and freeze-drying by a freeze-drying program, wherein the buffer solution comprises the following components: 4-morpholine ethanesulfonic acid, NaCl, ZnCl 2 、MgCl 2 ·6H 2 o, bovine serum albumin, casein, glycine, polyethylene glycol and trehalose. The brain specific protein product 9.5 can be effectively preserved, the activity of the freeze-dried product can be effectively preserved after the freeze-dried product is re-melted, and the detection sensitivity is improved; the freeze-drying protection method has a good preservation effect on the preservation of the brain specific protein product 9.5, and can ensure that the storage period of the PGP9.5 at 4-8 ℃ reaches 18 months and the storage period at-20 ℃ reaches 3 years.)

1. A method of lyophilizing a brain specific protein product 9.5, comprising the steps of: preparing a brain specific protein product 9.5 and a buffer solution into a solution, and performing freeze-drying by a freeze-drying program, wherein the buffer solution comprises the following components: 4-morpholine ethanesulfonic acid, NaCl, ZnCl₂、MgCl₂·6H₂o, bovine serum albumin, casein, glycine, polyethylene glycol and trehalose.

2. The lyophilization process according to claim 1, wherein the buffer comprises the following components in parts by weight: 5-30 parts of 4-morpholine ethanesulfonic acid, 8-12 parts of NaCl and ZnCl₂0.01-0.5 part of MgCl₂·6H₂0.1-15 parts of o, 2-50 parts of bovine serum albumin, 2-50 parts of casein, 1-15 parts of glycine, 0.2-25 parts of polyethylene glycol and 10-100 parts of trehalose.

3. The lyophilization process according to claim 1, wherein the buffer comprises the following components in parts by weight: 10-20 parts of 4-morpholine ethanesulfonic acid, 9-10 parts of NaCl and ZnCl₂0.01-0.1 part of MgCl₂·6H₂0.1-5 parts of o, 5-10 parts of bovine serum albumin, 5-10 parts of casein, 1-5 parts of glycine, 0.2-5 parts of polyethylene glycol and 10-30 parts of trehalose.

4. The lyophilization method according to claim 1, wherein the buffer further comprises a surfactant, wherein the surfactant is any one or more of tween 20, tween 60 or tween 80, preferably tween 20.

5. The lyophilization process of claim 1 wherein the buffer has a pH of 5.8 to 7.5.

6. The lyophilization process of claim 1, comprising the steps of: weighing surfactant, adding into purified water, and sequentially adding 4-morpholine ethanesulfonic acid, NaCl, and ZnCl₂、MgCl₂·6H₂And o, bovine serum albumin, casein, glycine, polyethylene glycol and trehalose, stirring until the bovine serum albumin, the casein, the glycine, the polyethylene glycol and the trehalose are completely dissolved, adjusting the pH value, fixing the volume and filtering.

7. The lyophilization process according to claim 6, wherein the surfactant is present in the buffer in a volume fraction of 0.02 to 1%; the filtration is carried out using a 0.2-0.5 μm filter, preferably a 0.22 μm filter.

8. The lyophilization process of claim 1, comprising the steps of:

(1) a pre-freezing section: placing a brain-specific protein product 9.5 into the buffer solution of any one of claims 1-5 at normal temperature to prepare a buffer solution system containing the brain-specific protein product 9.5, placing the buffer solution system into a sample cabin, firstly cooling the buffer solution system to 4 ℃, and preserving heat; cooling the buffer solution system to-40 ℃, preserving heat, cooling the buffer solution system to-55 ℃, and preserving heat;

(2) a sublimation drying section: reducing the vacuum degree of the sample chamber to 0-5 pa; then the temperature is raised to-40 ℃, and the temperature is preserved; then heating to-30 ℃, and preserving heat; then heating to-20 ℃, and preserving heat; then heating to-10 ℃, and preserving heat; then heating to-5 ℃, and preserving heat;

(3) a resolving and drying section: maintaining the vacuum degree of the sample chamber at 0-5 pa; setting the temperature end point to be 25 ℃, and raising the temperature from-5 ℃ to 25 ℃; the incubation was continued at 25 ℃.

9. The lyophilization process of claim 8,

step (1), the pre-freezing section,

cooling to 4 deg.C at normal temperature at a rate of 1-5 deg.C/min, and maintaining at 4 deg.C for 10-30 min;

cooling to-40 deg.C at 4 deg.C at a rate of 0.4-1 deg.C/min, and maintaining at-40 deg.C for 40-80 min;

heating to-20 deg.C at-40 deg.C at a heating rate of 0.5-1 deg.C/min, and maintaining at-20 deg.C for 30-60 min;

in the process of cooling from-20 ℃ to-55 ℃, the cooling rate is 0.25-0.5 ℃/min, the temperature is kept at-55 ℃ for 100-150min,

a step (2) of subjecting the mixture to sublimation drying,

heating at-55 deg.C to-40 deg.C at a heating rate of 0.5-1 deg.C/min, and maintaining at-40 deg.C for 40-80 min;

heating at-40 deg.C to-25 deg.C at a heating rate of 0.2-0.5 deg.C/min, and maintaining at-25 deg.C for 80-100 min;

in the process of heating from-25 ℃ to-15 ℃, the heating rate is 0.25-0.5 ℃/min, and the temperature is kept at-15 ℃ for 100 and 150 min;

in the process of heating from-15 ℃ to-5 ℃, the heating rate is 0.10-0.25 ℃/min, and the heat preservation is carried out at-5 ℃ for 100-150 min;

in the step (3), the sample is lifted to 25 ℃ from-5 ℃ in the desorption drying section at the temperature rise rate of 5-10 ℃/h; the continuous heat preservation time is 60-360 minutes at 25 ℃.

10. The lyophilization process of claim 8,

in the pre-freezing stage, the temperature is kept at 4 ℃ for 10-30 min; keeping the temperature at-40 deg.C for 60 min; keeping the temperature at-20 ℃ for 30 min; keeping the temperature at-55 deg.C for 120 min;

step (2), the sublimation drying section is subjected to heat preservation for 60min at the temperature of minus 40 ℃; keeping the temperature at-25 ℃ for 90 min; keeping the temperature at-15 deg.C for 120 min; keeping the temperature at-5 ℃ for 120 min.

Technical Field

The invention belongs to the technical field of preservation of a brain specific protein product 9.5, and particularly relates to a buffer solution and a freeze-drying method of the brain specific protein product 9.5.

Background

The ubiquitin-proteasome pathway is an important pathway for regulating intracellular protein activity. The main subjects for the regulation are functional proteins with damaged intracellular structures or need to be cleared in time. The regulation of the ubiquitin-proteasome pathway is closely related to many processes such as cell cycle, signal transduction, developmental differentiation, substance metabolism and the like, and the ubiquitin-proteasome system is considered to realize the rapid and accurate regulation and control of target proteins mainly through ubiquitination enzymes and deubiquitinase at present.

The brain specific protein product 9.5(protein gene product 9.5, PGP9.5), also known as Ubiquitin carboxyl-terminal hydrolase (UCH-L1), is a cysteine hydrolase consisting of 223 amino acids with a molecular weight of 24800 Da. UCH-L1 will cross the blood brain barrier into the blood within 1 hour after TBI occurs, resulting in a significant elevation of serum UCH-L1. Has important significance for early diagnosis, differential diagnosis and prognosis judgment of TBI, and is mainly used for auxiliary diagnosis of brain trauma clinically. The use of the early biomarker ubiquitin carboxy-terminal hydrolase L1(UCH-L1) as disclosed in chinese patent application CN201880020489.5 aids in the diagnosis and evaluation of human subjects who have suffered or may have suffered damage to the head, such as mild or moderate to severe Traumatic Brain Injury (TBI). Also disclosed herein are methods that facilitate determining whether a subject who has suffered or is likely to have suffered damage to the head will benefit from, and therefore receive, a head Computed Tomography (CT) scan based on the level of UCH-L1. These methods involve detecting changes in levels of UCH-L1 and levels of UCH-L1 in one or more samples taken from a human subject at a time point within 24 hours after the subject has suffered or may have suffered damage to the head.

Therefore, the preservation of ubiquitin carboxyl-terminal hydrolase has extremely important significance. However, there is no targeted preservation method in the prior art, and the preservation effect of ubiquitin carboxyl terminal hydrolase cannot be improved well in the prior art, and the activity, detection sensitivity and other performances after re-fusion are affected by different degrees.

The invention aims to provide a freeze-drying method of a brain-specific protein product 9.5, so as to improve the preservation activity of the brain-specific protein product 9.5 and better develop a detection technology of the brain-specific protein product 9.5.

Disclosure of Invention

In order to overcome the technical problems, the invention provides a buffer solution which can effectively improve the activity of PGP9.5, improve the detection sensitivity of an antigen freeze-dried product and simultaneously effectively prolong the storage life of the PGP 9.5.

In order to achieve the above purpose, the technical scheme provided by the invention is as follows:

a method of lyophilizing a brain specific protein product 9.5, comprising the steps of: preparing a brain specific protein product 9.5 and a buffer solution into a solution, and performing freeze-drying by a freeze-drying program, wherein the buffer solution comprises the following components: 4-Morpholinoethanesulfonic acid, NaCl (sodium chloride), ZnCl₂(Zinc chloride), MgCl₂·6H₂o (magnesium chloride hexahydrate), bovine serum albumin, casein, glycine, polyethylene glycol, trehalose.

Preferably, the buffer solution comprises the following components in parts by weight: 5-30 parts of 4-morpholine ethanesulfonic acid, 8-12 parts of NaCl and ZnCl₂0.01-0.5 part of MgCl₂·6H₂0.1-15 parts of o, 2-50 parts of bovine serum albumin, 2-50 parts of casein, 1-15 parts of glycine, 0.2-25 parts of polyethylene glycol and 10-100 parts of trehalose.

Preferably, the buffer solution comprises the following components in parts by weight: 10-20 parts of 4-morpholine ethanesulfonic acid, 9-10 parts of NaCl and ZnCl₂0.01-0.1 part of MgCl₂·6H₂o 0.1-5 parts, bovine serum albumin 5-10 parts, casein 5-10 parts, glycine 1-5 parts, polyethylene glycol 0.2-5 parts, seaweed10-30 parts of sugar.

Preferably, the buffer solution further comprises a surfactant.

Preferably, the surfactant is any one or more of tween 20, tween 60 or tween 80, preferably tween 20;

preferably, the pH of the buffer is between 5.8 and 7.5.

Another object of the present invention is to provide a method for preparing the buffer solution, comprising the steps of:

weighing surfactant, adding into purified water, and sequentially adding 4-morpholine ethanesulfonic acid, NaCl, and ZnCl₂、MgCl₂·6H₂And o, bovine serum albumin, casein, glycine, polyethylene glycol and trehalose, stirring until the bovine serum albumin, the casein, the glycine, the polyethylene glycol and the trehalose are completely dissolved, adjusting the pH value, fixing the volume and filtering.

Preferably, the surfactant is present in the buffer in a volume fraction of 0.02 to 1%.

Preferably, the filtration is performed using a 0.2-0.5 μm filter, preferably a 0.22 μm filter.

It is also an object of the present invention to provide the use of said buffer for the preservation of a brain specific protein product 9.5.

It is also an object of the present invention to provide a method for lyophilizing a brain-specific protein product 9.5, comprising the steps of:

(1) a pre-freezing section: at normal temperature, putting the brain specific protein product 9.5 into a buffer solution to prepare a buffer solution system containing the brain specific protein product 9.5, putting the buffer solution system into a sample cabin, firstly cooling the buffer solution system to 4 ℃, and preserving heat; cooling the buffer solution system to-40 ℃, preserving heat, heating the buffer solution system to-20 ℃, preserving heat, cooling the buffer solution system to-55 ℃, and preserving heat;

(2) a sublimation drying section: reducing the vacuum degree of the sample chamber to 0-5 pascal (pa); then the temperature is raised to-40 ℃, and the temperature is preserved; then heating to-25 ℃, and preserving heat; then heating to-15 ℃, and preserving heat; then heating to-5 ℃, and preserving heat;

(3) a resolving and drying section: maintaining the vacuum degree of the sample chamber at 0-5 pa; setting the temperature end point to be 25 ℃, and raising the temperature from-5 ℃ to 25 ℃; the incubation was continued at 25 ℃.

Preferably, step (1), said pre-freezing section,

in the process of cooling to 4 ℃ at normal temperature, the cooling rate is 1-5 ℃/min, and the temperature is kept at 4 ℃ for 10-30min, preferably 30 min; (ii) a

Cooling to-40 deg.C at 4 deg.C at a rate of 0.4-1 deg.C/min, and maintaining at-40 deg.C for 40-80 min; preferably 60 min;

heating to-20 deg.C at-40 deg.C at a heating rate of 0.5-1 deg.C/min, and maintaining at-20 deg.C for 30-60 min; preferably 30 min; in the process of cooling from-20 ℃ to-55 ℃, the cooling rate is 0.25-0.5 ℃/min, and the temperature is kept at-55 ℃ for 100-150min, preferably 120 min;

preferably, step (2), the sublimation drying section,

heating at-55 deg.C to-40 deg.C at a heating rate of 0.5-1 deg.C/min, and maintaining at-40 deg.C for 40-80min, preferably 60 min;

heating to-25 deg.C at-40 deg.C at a heating rate of 0.2-0.5 deg.C/min, and maintaining at-25 deg.C for 80-100min, preferably 90 min;

in the process of heating from-25 ℃ to-15 ℃, the heating rate is 0.25-0.5 ℃/min, and the temperature is kept at-15 ℃ for 100 and 150min, preferably 120 min;

in the process of heating from-15 ℃ to-5 ℃, the heating rate is 0.10-0.25 ℃/min, and the temperature is kept at-5 ℃ for 100 and 150min, preferably 120 min;

preferably, in the step (3), the desorption drying section raises the sample from-5 ℃ to 25 ℃ at a temperature raising rate of 5-10 ℃/h; the continuous heat preservation time is 60-360 minutes at 25 ℃.

Compared with the prior art, the invention has the technical advantages that:

(1) the invention provides a method for preserving a brain specific protein product 9.5, which can effectively preserve the brain specific protein product 9.5, effectively preserve the activity of a compression-resistant freeze-dried product in the detection process of the brain specific protein product 9.5 after re-melting, and improve the detection sensitivity.

(2) The freeze-drying protection method provided by the invention has a good preservation effect on the preservation of the brain specific protein product 9.5, and can ensure that the storage period of the PGP9.5 at 4-8 ℃ reaches 18 months and the storage period at-20 ℃ reaches 3 years.

(3) The components of the buffer solution, such as 4-morpholine ethanesulfonic acid, glycine, polyethylene glycol, trehalose and the like, have good synergistic effect, can effectively improve the preservation effect of the buffer solution on the brain specific protein product 9.5, and improve the biological activity.

Detailed Description

The present invention will be described below with reference to specific examples to make the technical aspects of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

Example 1

A method of lyophilizing a brain specific protein product 9.5, comprising the steps of: preparing a brain specific protein product 9.5 and a buffer solution into a solution, and performing freeze-drying by a freeze-drying program, wherein the buffer solution comprises the following components: comprises the following components: 10g of 4-morpholine ethanesulfonic acid, 9g of NaCl and ZnCl₂ 0.1g、MgCl₂·6H₂o 0.1g, bovine serum albumin 10g, casein 5g, glycine 10g, polyethylene glycol 0.2g, trehalose 20g, tween 205 ml.

The preparation method of the buffer solution comprises the following steps: weighing surfactant, adding into purified water, and sequentially adding 4-morpholine ethanesulfonic acid, NaCl, and ZnCl₂、MgCl₂·6H₂o, bovine serum albumin, casein, glycine, polyethylene glycol and trehalose, stirring until the bovine serum albumin, the casein, the glycine, the polyethylene glycol and the trehalose are completely dissolved, adjusting the pH value to 6.5, fixing the volume to 1000ml, and filtering by using a filter membrane of 0.22 mu m.

A method of lyophilizing the brain specific protein product 9.5, comprising the steps of:

(1) a pre-freezing section: at normal temperature, putting the brain specific protein product 9.5 into a buffer solution to prepare a buffer solution system containing the brain specific protein product 9.5, putting the buffer solution system into a sample cabin, firstly cooling the buffer solution system to 4 ℃, and preserving heat; cooling the buffer solution system to-40 ℃, preserving heat, heating the buffer solution system to-20 ℃, preserving heat, cooling the buffer solution system to-55 ℃, and preserving heat;

(2) a sublimation drying section: adjusting the vacuum degree of the sample chamber; then the temperature is raised to-40 ℃, and the temperature is preserved; then heating to-25 ℃, and preserving heat; then heating to-15 ℃, and preserving heat; then heating to-5 ℃, and preserving heat;

(3) a resolving and drying section: adjusting the vacuum degree of the sample chamber; setting a temperature end point to be 25 ℃; keeping the temperature at 25 ℃;

lyophilization procedure, table 1 below:

table 1 lyophilization procedure of example 1

Example 2

A method of lyophilizing a brain specific protein product 9.5, comprising the steps of: preparing a brain specific protein product 9.5 and a buffer solution into a solution, and performing freeze-drying by a freeze-drying program, wherein the buffer solution comprises the following components: 20g of 4-morpholine ethanesulfonic acid, 10g of NaCl and ZnCl₂ 0.01g、MgCl₂·6H₂o 5g, bovine serum albumin 5g, casein 10g, glycine 1g, polyethylene glycol 5g, trehalose 10g, and tween 600.2 ml.

The preparation method of the buffer solution comprises the following steps: weighing surfactant, adding into purified water, and sequentially adding 4-morpholine ethanesulfonic acid, NaCl, and ZnCl₂、MgCl₂·6H₂o, bovine serum albumin, casein, glycine, polyethylene glycol and trehalose, stirring until the bovine serum albumin, the casein, the glycine, the polyethylene glycol and the trehalose are completely dissolved, adjusting the pH value to 5.8, fixing the volume to 1000ml, and filtering by using a filter membrane of 0.22 mu m.

A method of lyophilizing the brain specific protein product 9.5, comprising the steps of:

(1) a pre-freezing section: at normal temperature, putting the brain specific protein product 9.5 into a buffer solution to prepare a buffer solution system containing the brain specific protein product 9.5, putting the buffer solution system into a sample cabin, firstly cooling the buffer solution system to 4 ℃, and preserving heat; cooling the buffer solution system to-40 ℃, preserving heat, heating the buffer solution system to-20 ℃, preserving heat, cooling the buffer solution system to-55 ℃, and preserving heat;

(2) a sublimation drying section: adjusting the vacuum degree of the sample chamber; then the temperature is raised to-40 ℃, and the temperature is preserved; then heating to-25 ℃, and preserving heat; then heating to-15 ℃, and preserving heat; then heating to-5 ℃, and preserving heat;

(3) a resolving and drying section: adjusting the vacuum degree of the sample chamber; setting a temperature end point to be 25 ℃; keeping the temperature at 25 ℃;

lyophilization procedure, table 2 below:

table 2 lyophilization procedure of example 2

Example 3

A method of lyophilizing a brain specific protein product 9.5, comprising the steps of: preparing a brain specific protein product 9.5 and a buffer solution into a solution, and performing freeze-drying by a freeze-drying program, wherein the buffer solution comprises the following components: 5g of 4-morpholine ethanesulfonic acid, 8g of NaCl and ZnCl₂ 0.5g、MgCl₂·6H₂o 0.1g, bovine serum albumin 2g, casein 15g, glycine 5g, polyethylene glycol 0.2g, trehalose 30g, tween 8010 ml.

The preparation method of the buffer solution comprises the following steps: weighing surfactant, adding into purified water, and sequentially adding 4-morpholine ethanesulfonic acid, NaCl, and ZnCl₂、MgCl₂·6H₂o, bovine serum albumin, casein, glycine, polyethylene glycol and trehalose, stirring until the bovine serum albumin, the casein, the glycine, the polyethylene glycol and the trehalose are completely dissolved, adjusting the pH value to 7.5, fixing the volume to 1000ml, and filtering by using a filter membrane of 0.45 mu m.

A method of lyophilizing the brain specific protein product 9.5, comprising the steps of:

(1) a pre-freezing section: at normal temperature, putting the brain specific protein product 9.5 into a buffer solution to prepare a buffer solution system containing the brain specific protein product 9.5, putting the buffer solution system into a sample cabin, firstly cooling the buffer solution system to 4 ℃, and preserving heat; cooling the buffer solution system to-40 ℃, preserving heat, heating the buffer solution system to-20 ℃, preserving heat, cooling the buffer solution system to-55 ℃, and preserving heat;

(2) a sublimation drying section: adjusting the vacuum degree of the sample chamber; then the temperature is raised to-40 ℃, and the temperature is preserved; then heating to-25 ℃, and preserving heat; then heating to-15 ℃, and preserving heat; then heating to-5 ℃, and preserving heat;

(3) a resolving and drying section: adjusting the vacuum degree of the sample chamber; setting a temperature end point to be 25 ℃; keeping the temperature at 25 ℃;

lyophilization procedure, table 3 below:

table 3 lyophilization procedure of example 3

Example 4

A method of lyophilizing a brain specific protein product 9.5, comprising the steps of: preparing a brain specific protein product 9.5 and a buffer solution into a solution, and performing freeze-drying by a freeze-drying program, wherein the buffer solution comprises the following components: 10g of 4-morpholine ethanesulfonic acid, 12g of NaCl and ZnCl₂ 0.01g、MgCl₂·6H₂o 10g, bovine serum albumin 13g, casein 2g, glycine 1g, polyethylene glycol 7g, trehalose 10g and tween 205 ml.

The preparation method of the buffer solution comprises the following steps: weighing surfactant, adding into purified water, and sequentially adding 4-morpholine ethanesulfonic acid, NaCl, and ZnCl₂、MgCl₂·6H₂o, bovine serum albumin, casein, glycine, polyethylene glycol and trehalose, stirring until the bovine serum albumin, the casein, the glycine, the polyethylene glycol and the trehalose are completely dissolved, adjusting the pH value to 7.0, fixing the volume to 1000ml, and filtering by using a filter membrane of 0.22 mu m.

A method of lyophilizing the brain specific protein product 9.5, comprising the steps of:

(1) a pre-freezing section: at normal temperature, putting the brain specific protein product 9.5 into a buffer solution to prepare a buffer solution system containing the brain specific protein product 9.5, putting the buffer solution system into a sample cabin, firstly cooling the buffer solution system to 4 ℃, and preserving heat; cooling the buffer solution system to-40 ℃, preserving heat, heating the buffer solution system to-20 ℃, preserving heat, cooling the buffer solution system to-55 ℃, and preserving heat;

(2) a sublimation drying section: adjusting the vacuum degree of the sample chamber; then the temperature is raised to-40 ℃, and the temperature is preserved; then heating to-25 ℃, and preserving heat; then heating to-15 ℃, and preserving heat; then heating to-5 ℃, and preserving heat;

(3) a resolving and drying section: adjusting the vacuum degree of the sample chamber; setting a temperature end point to be 25 ℃; keeping the temperature at 25 ℃;

lyophilization procedure, table 4 below:

table 4 example 4 lyophilization procedure

Comparative example 1

The difference compared to example 1 is that 4-morpholinoethanesulfonic acid is replaced with tris.

A method of lyophilizing a brain specific protein product 9.5, comprising the steps of: preparing a brain specific protein product 9.5 and a buffer solution into a solution, and performing freeze-drying by a freeze-drying program, wherein the buffer solution comprises the following components: comprises the following components: 10g of tris (hydroxymethyl) aminomethane, 9g of NaCl, and ZnCl₂ 0.1g、MgCl₂·6H₂o 0.1g, bovine serum albumin 10g, casein 5g, glycine 10g, polyethylene glycol 0.2g, trehalose 20g, tween 205 ml.

The preparation method of the buffer solution comprises the following steps: weighing surfactant, adding into purified water, sequentially adding tris (hydroxymethyl) aminomethane, NaCl and ZnCl₂、MgCl₂·6H₂o, bovine serum albumin, casein, glycine, polyethylene glycol and trehalose, stirring until the bovine serum albumin, the casein, the glycine, the polyethylene glycol and the trehalose are completely dissolved, adjusting the pH value to 6.5, fixing the volume to 1000ml, and filtering by using a filter membrane of 0.22 mu m.

The lyophilization process for the brain specific protein product 9.5 was the same as in example 1.

Comparative example 2

The difference compared to example 1 is that trehalose was replaced by sucrose.

A method of lyophilizing a brain specific protein product 9.5, comprising the steps of: preparing a brain specific protein product 9.5 and a buffer solution into a solution, and performing freeze-drying by a freeze-drying program, wherein the buffer solution comprises the following components: comprises the following components: 10g of 4-morpholine ethanesulfonic acid, 9g of NaCl and ZnCl₂ 0.1g、MgCl₂·6H₂o 0.1g, bovine serum albumin 10g, casein 5g, glycine 10g, polyethylene glycol 0.2g, sucrose 20g, and tween 205 ml.

The preparation method of the buffer solution comprises the following steps: weighing surfactant, adding into purified water, and sequentially adding 4-morpholine ethanesulfonic acid, NaCl, and ZnCl₂、MgCl₂·6H₂o, bovine serum albumin, casein, glycine, polyethylene glycol and sucrose, stirring until the bovine serum albumin, the casein, the glycine, the polyethylene glycol and the sucrose are completely dissolved, adjusting the pH value to 6.5, fixing the volume to 1000ml, and filtering by using a filter membrane of 0.22 mu m.

The lyophilization process for the brain specific protein product 9.5 was the same as in example 1.

Comparative example 3

The difference compared to example 1 is that glycine is replaced by glycerol.

A method of lyophilizing a brain specific protein product 9.5, comprising the steps of: preparing a brain specific protein product 9.5 and a buffer solution into a solution, and performing freeze-drying by a freeze-drying program, wherein the buffer solution comprises the following components: comprises the following components: 10g of 4-morpholine ethanesulfonic acid, 9g of NaCl and ZnCl₂ 0.1g、MgCl₂·6H₂o 0.1g, bovine serum albumin 10g, casein 5g, glycerol 10g, polyethylene glycol 0.2g, trehalose 20g, and tween 205 ml.

The preparation method of the buffer solution comprises the following steps: weighing surfactant, adding into purified water, and sequentially adding 4-morpholine ethanesulfonic acid, NaCl, and ZnCl₂、MgCl₂·6H₂o, bovine serum albumin, casein, glycerol, polyethylene glycol and trehalose, stirring to completely dissolve, adjusting pH to 6.5 and fixing volume to 1000ml, and usingFiltering with 0.22 μm filter membrane.

The lyophilization process for the brain specific protein product 9.5 was the same as in example 1.

Comparative example 4

Compared with example 1, with the difference that MgCl is used₂·6H₂o is replaced by enzymatic hydrolysis of gelatin.

A method of lyophilizing a brain specific protein product 9.5, comprising the steps of: preparing a brain specific protein product 9.5 and a buffer solution into a solution, and performing freeze-drying by a freeze-drying program, wherein the buffer solution comprises the following components: comprises the following components: 10g of 4-morpholine ethanesulfonic acid, 9g of NaCl and ZnCl₂0.1g, 0.1g of enzymatically hydrolyzed gelatin, 10g of bovine serum albumin, 5g of casein, 10g of glycine, 0.2g of polyethylene glycol, 20g of trehalose and 205 ml of tween.

The preparation method of the buffer solution comprises the following steps: weighing surfactant, adding into purified water, and sequentially adding 4-morpholine ethanesulfonic acid, NaCl, and ZnCl₂Hydrolyzing gelatin, bovine serum albumin, casein, glycine, polyethylene glycol and trehalose by an enzyme method, stirring until the gelatin, bovine serum albumin, casein, glycine, polyethylene glycol and trehalose are completely dissolved, adjusting the pH value to 6.5, fixing the volume to 1000ml, and filtering by using a filter membrane of 0.22 mu m.

The lyophilization process for the brain specific protein product 9.5 was the same as in example 1.

Comparative example 5

The difference is in the temperature rise program, compared to example 1.

A method of lyophilizing a brain specific protein product 9.5, comprising the steps of:

(1) a pre-freezing section: at normal temperature, putting the brain specific protein product 9.5 into a buffer solution to prepare a buffer solution system containing the brain specific protein product 9.5, putting the buffer solution system into a sample cabin, firstly cooling the buffer solution system to 4 ℃, and preserving heat; cooling the buffer solution system to-40 ℃, preserving heat, cooling the buffer solution system to-55 ℃, and preserving heat;

(2) a sublimation drying section: adjusting the vacuum degree of the sample chamber; then the temperature is raised to-40 ℃, and the temperature is preserved; then heating to-30 ℃, and preserving heat; then heating to-20 ℃, and preserving heat; then heating to-10 ℃, and preserving heat; then heating to-5 ℃, and preserving heat;

(3) a resolving and drying section: adjusting the vacuum degree of the sample chamber; setting a temperature end point to be 25 ℃; keeping the temperature at 25 ℃; the lyophilization procedure, among others, is shown in the following table:

table 5 comparative example 5 lyophilization procedure

Evaluation of Effect

Evaluation of test Effect

Using the buffers prepared in examples 1 to 4 and comparative examples 1 to 5, lyophilizates of PGP9.5 were prepared at various concentrations and lyophilized. The related detection of the immunoreaction is carried out by adopting a full-automatic chemiluminescence immunoassay analyzer self-developed by Beijing America Thailand Biotechnology Limited.

Detecting the index

1. Water content

5 bottles of lyophilized products with the concentrations of 160pg/mL and 1280pg/mL are taken. The freeze-dried product was weighed after removing the cap and plug and recorded as M1_n(n is 1, 2, 3 … … 10). Drying at 200 deg.C until the mass no longer changes, weighing again, and recording as M2_n(n is 1, 2, 3 … … 10). The vials containing the sample were washed clean in sequence, oven dried at 200 ℃ until the mass no longer changed, and the vials were weighed in sequence and recorded as M3_n(n is 1, 2, 3 … … 10). And calculating the water content according to a formula. The water content of the lyophilized product should be less than 2%.

Water content [ (M1)_n-M3_n)/(M2_n-M3_n)-1]×100％

The average water content of the corresponding lyophilizates of examples 1 to 4 and comparative example 5 was tested using the method described above, and the results are shown in Table 6.

TABLE 6 Water content

Test group	Water content%
		Example 1	0.59
Example 2	1.15
		Example 3	0.99
Example 4	1.42
		Comparative example 5	5.24

2. Accuracy of

A solution (A) of brain-specific protein product 9.5(PGP9.5) at a concentration of about 3000pg/mL (tolerance. + -. 10%) was added to a serum sample (B) at a concentration ranging from 0pg/mL to 80pg/mL, at a volume ratio of 1:9 between the added PGP9.5 antigen and the sample B, and the assay was carried out using a lyophilizate and a kit for detecting brain-specific protein product 9.5 (magnetoparticle chemiluminescence). The recovery rate R is calculated according to the following formula and should be in the range of 85-115%.

In the formula: r is the recovery rate; v is the volume of the sample A liquid; v₀Is the volume of serum sample B; c is the average value of 3 measurements after the serum sample B liquid is added into the A liquid; c₀The average value of 3 measurements of the serum sample B liquid is shown; c_SIs as followsConcentration of the product A solution.

3. Linear interval

High value samples near the upper limit of the linear zone were mixed with low value samples or zero concentration samples near the lower limit of the linear zone to 5 concentrations of 160pg/mL, 320pg/mL, 640pg/mL, 1280pg/mL, 2560pg/mL, respectively. The test was repeated 3 times for each concentration of the sample to obtain the luminescence value, the measurement result of each sample was recorded, and the average value (y) of the 3 measurements of each sample was calculated_i). In diluted concentration (x)_i) As independent variable, the mean value (y) of the results is determined_i) Linear regression equations were solved for the dependent variables.

The correlation coefficient (r) of the linear regression is calculated according to the following formula, and the correlation coefficient r should be more than or equal to 0.990 in the linear interval of [80,2560] pg/mL.

In the formula: r is a correlation coefficient; x is the number of_iIs a dilution ratio; y is_iDetermining a mean value for each sample;is the average of the dilution ratios;is the overall mean value of the sample measurement results.

4. Repeatability of

The freeze-dried product with the concentration of 320pg/mL is repeatedly tested for 10 times, and the average value of the 10 test results is calculatedAnd standard deviation SD.

The Coefficient of Variation (CV) was calculated as follows, and as a result, CV should be less than or equal to 8%.

In the formula: s is the standard deviation of the sample test values；Is the average of the sample test values.

5. Sensitivity of the probe

Repeating the test for 20 times to obtain concentration values of 20 test results, and calculating average valueAnd Standard Deviation (SD). Mean value ofThe result is less than 80 pg/mL.

6. Difference between batches

The quality control material with a concentration of 640pg/mL was tested 10 times in duplicate with 3 lot kits, respectively. The average of the results of 30 tests was calculatedAnd standard deviation SD, and obtaining Coefficient of Variation (CV) according to the following formula, wherein the result is less than or equal to 15%.

In the formula: s is the standard deviation of the sample test values;is the average of the sample test values.

The performance indexes of freeze-dried products which are freeze-dried and stored for 0 month, 18 months, 20 months, 36 months and 39 months are evaluated by adopting the method, and the results are as follows.

TABLE 7 test data (Normal temperature 0 month)

TABLE 8 stability effect data (4-8 ℃ C. for 18 months)

Test group	Linear interval r	Accuracy%	Repetitive CV	Sensitivity (pg/mL)
					Example 1	0.9961	88.56	6.27	62.99
Example 2	0.9958	87.61	8.61	76.90
					Example 3	0.9962	91.93	8.50	70.48
Example 4	0.9955	94.14	4.39	75.88
					Comparative example 1	0.9632	83.41	20.72	190.22
Comparative example 2	0.9638	84.67	16.77	177.58
					Comparative example 3	0.9725	81.58	17.59	191.85
Comparative example 4	0.9693	83.63	22.53	128.19
					Comparative example 5	0.9673	74.67	19.29	205.74

TABLE 9 stability effect data (4-8 ℃ C. for 20 months)

TABLE 10 stability Effect data (-20 ℃ C. for 36 months)

Test group	Linear interval r	Accuracy%	Repetitive CV	Sensitivity (pg/mL)
					Example 1	0.9973	87.88	7.26	65.06
Example 2	0.9906	85.47	6.68	70.31
					Example 3	0.9915	86.69	7.33	64.69
Example 4	0.9932	88.81	7.15	77.73
					Comparative example 1	0.9854	78.33	11.95	106.67
Comparative example 2	0.9796	84.08	13.67	129.84
					Comparative example 3	0.9826	79.57	19.36	149.01
Comparative example 4	0.9722	75.04	14.64	170.61
					Comparative example 5	0.9759	83.35	14.68	151.95

TABLE 11 stability Effect data (-20 ℃ C. for 39 months)

Therefore, the buffer solution and the freeze-drying method provided by the invention can effectively store PGP9.5, can effectively store the activity of the anti-compression freeze-dried product after re-melting in the detection process of the glial fibrillary acidic protein, and improve the detection sensitivity.

The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.