CN112485451B - Interleukin 6 freeze-drying calibrator, preparation method thereof and freeze-drying protective solution - Google Patents
Interleukin 6 freeze-drying calibrator, preparation method thereof and freeze-drying protective solution Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, and particularly discloses an interleukin 6 freeze-drying calibrator, a preparation method thereof and a freeze-drying protective solution. The freeze-drying protective solution comprises the following components: buffer solution, trehalose, gamma-cyclodextrin, mannitol, a collagen protective agent, a surfactant and a preservative, wherein the pH value of the freeze-drying protective solution is 7.2-7.6; the freeze-drying protective liquid can stably maintain the properties of the interleukin 6 calibrator, has good toughness and good re-solubility, can also protect the activity of the interleukin 6 calibrator to the greatest extent, and prolongs the storage time of the interleukin 6 calibrator.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to an interleukin 6 freeze-drying calibrator, a preparation method thereof and a freeze-drying protective solution.
Background
Interleukin-6 (IL-6) is a kind of cytokine, it is a kind of interleukin, can stimulate the cell that participates in the immune response to proliferate, differentiate and synthesize and improve its function, activated lymphocyte, monocyte macrophage, bone marrow cell and some tumor cells secrete IL-6, IL-6 is produced in the immune response as an organism's important medium, have many biological activities.
At present, most of calibrators in domestic interleukin 6(IL-6) detection kits are liquid, IL-6 antigens are easy to denature in the liquid state, therefore, the preservation time of the IL-6 antigen in the liquid state can be prolonged by adding some protective agents (such as some proteins, saccharides, antioxidants and the like), for example, Chinese patent document CN110988367A discloses a storage agent, a calibrator for detecting LH and a detection kit, wherein the storage agent comprises a buffer solution, 8-aniline-1-naphthalene sulfonic acid and/or salt thereof, a protective agent and a surfactant, the storage agent can stably store a plurality of antigens and prolong the storage life of the solution, but the shelf life of the liquid IL-6 calibrator is generally 1-2 weeks when the liquid IL-6 calibrator is placed at 2-8 ℃ in the presence of a protective agent, the storage time is relatively short, and the validity period of an IL-6 detection kit is limited.
In addition, the liquid IL-6 calibrator is generally stored at 2-8 ℃, and needs to be transported by using ice bags or cold chains during transportation, so that the cost of the transportation process is increased, and meanwhile, the liquid state has leakage risk during the transportation process; and the liquid IL-6 antigen is only stable under higher concentration, the shelf life of the high-concentration IL-6 antigen is 6 months when the preservation temperature is minus 80 ℃, and the shelf life of the high-concentration IL-6 antigen is 3 months when the preservation temperature is minus 20 ℃, so that the shelf life is shorter.
Therefore, a freeze-drying preservation solution and a method for preparing an interleukin 6 freeze-drying calibrator by using the freeze-drying preservation solution are needed.
Disclosure of Invention
The freeze-drying protective solution prepared by the invention furthest protects the activity of the interleukin 6 calibrator, prolongs the storage time of the interleukin 6 calibrator, can maintain the freeze-dried property of the interleukin 6 calibrator, and has the advantages of no sticking to bottles, no collapse, good toughness and good re-solubility.
In order to achieve the purpose, the invention adopts the technical scheme that:
a freeze-drying protective solution comprises the following components: buffer solution, trehalose, gamma-cyclodextrin, mannitol, a collagen protective agent, a surfactant and a preservative, wherein the pH value of the freeze-drying protective solution is 7.2-7.6.
Through a large amount of researches and experiments, the inventor discovers that the activity of the interleukin 6 calibrator can be effectively maintained by compounding various components of trehalose, a collagen protective agent, mannitol and gamma-cyclodextrin, the activity is basically kept unchanged before and after freeze-drying, the stability of the interleukin 6 calibrator is obviously improved, the transportation of freeze-dried interleukin 6 antigen is facilitated, the storage time of the interleukin 6 calibrator is prolonged, and the storage time of an interleukin 6 detection kit is prolonged; the interleukin 6 calibrator prepared by the freeze-drying protective solution is not shrunk after freeze-drying, has uniform and stable appearance and good redissolution property.
The buffer solution provides a stable pH environment for the freeze-drying protective solution, and avoids the influence on the activity of protein molecules caused by unstable pH due to weak buffer capacity; the trehalose can form a unique protective film on the cell surface under the conditions of high temperature, high cold, high osmotic pressure and dry water loss, and can effectively prevent the interleukin 6 protein molecules from being inactivated without denaturation; the mannitol contains a plurality of hydroxyl groups as a filling agent, can replace water to form hydrogen bonds with hydrophilic groups of the IL-6 antigen, better protects the spatial structure of the IL-6 antigen and has a moisturizing effect, improves the toughness of the interleukin 6 calibrator after freeze-drying, and is beneficial to maintaining the freeze-drying form of the interleukin 6 calibrator; the collagen protective agent is a high-purity pig dermis collagen polypeptide component, has good protein-stabilizing property, has good protective effect on protein antigens, and is easy to dissolve in water; the surfactant can prevent protein aggregation and can achieve the effect of solubilization; the preservative has the effect of inhibiting the growth of bacteria and mould and can improve the stability of the freeze-dried antigen after redissolution.
As a preferred embodiment of the freeze-drying protection solution, the freeze-drying protection solution comprises the following components in percentage by weight: 0.5-3% of trehalose, 0.5-5% of gamma-cyclodextrin, 0.5-5% of mannitol, 0.2-1% of collagen protective agent, 0.05-0.1% of surfactant, 0.05-0.1% of preservative and the balance of Tris-HCl buffer solution or HEPES-Na buffer solution, wherein the concentration of the Tris-HCl buffer solution or the HEPES-Na buffer solution is 10mM-50 mM. .
As a preferred embodiment of the freeze-drying protection solution, the freeze-drying protection solution comprises the following components in percentage by weight: 0.5% of trehalose, 1% of gamma-cyclodextrin, 1% of mannitol, 0.5% of collagen protective agent, 0.1% of surfactant, 0.1% of preservative and the balance of Tris-HCl buffer solution, wherein the concentration of the Tris-HCl buffer solution is 20 mM. Activity evaluation and stability evaluation show that the activity of the interleukin 6 calibrator treated by the freeze-drying protective solution is basically kept unchanged before and after freeze-drying, the activity difference with the activity before freeze-drying is within 5%, and the deviation of detection results of the interleukin 6 freeze-drying calibrator after being placed at 37 ℃ for 30 days and after being placed at 4 ℃ after being opened is within 5%, so that the freeze-drying protective solution can improve the stability of the interleukin 6 calibrator and prolong the storage time of the interleukin 6 calibrator and an interleukin 6 detection kit; the appearance of the interleukin 6 calibrator after freeze-drying is not shrunk, a bottle is not stuck, the toughness is good, the residual moisture is low, and the long-term storage is facilitated; and standing for 10s after adding purified water, wherein the interleukin 6 freeze-dried calibrator can be completely dissolved, and the solution is clear without block and foam.
As a preferred embodiment of the freeze-drying protective solution of the present invention, the preparation method of the collagen protective agent is as follows:
s1, extracting collagen by an enzymolysis method: cleaning animal skin, adding purified water, heating, taking out, cutting, degreasing, filtering to obtain filter residue, adding acetic acid, pepsin and compound protease, stirring, soaking, performing enzymolysis, centrifuging to obtain supernatant, adding polyethylene glycol 6000, performing collagen salting-out, centrifuging to obtain precipitate to obtain collagen;
s2, purifying collagen: heating the collagen prepared in the step S1 in a boiling water bath to inactivate enzymes, centrifuging to obtain supernatant, purifying the supernatant, and intercepting short peptides with molecular weight less than 5kd to obtain the collagen protective agent.
As a preferred embodiment of the freeze-drying protection solution, the enzyme activity of the pepsin and compound protease is 2000U/mL.
As a preferred embodiment of the freeze-drying protection solution of the present invention, the mannitol can better improve the toughness of the freeze-dried interleukin 6 calibrator, and is beneficial to maintaining the freeze-drying form of the interleukin 6 calibrator.
As a preferred embodiment of the freeze-drying protection solution, the surfactant is tween 80, and the preservative is KY 100.
The second purpose of the invention is to provide an interleukin 6 freeze-drying calibrator, which is obtained by mixing the interleukin 6 calibrator with the freeze-drying protective solution.
The third purpose of the invention is to provide a method for preparing an interleukin 6 freeze-drying calibrator by using the freeze-drying protective solution, which comprises the following steps:
A. mixing: mixing the interleukin 6 calibrator with the freeze-drying protective solution to obtain a mixture;
B. a pre-freezing stage: c, freezing the mixture prepared in the step A for 2-3 hours at the temperature of-35 to-25 ℃;
C. a sublimation drying stage: raising the temperature from-35 to-25 ℃ to-20 to-10 ℃, wherein the temperature raising time is 0.5 to 1 hour, and drying the mixture subjected to the pre-freezing stage for 10 to 12 hours at the temperature of-20 to-10 ℃;
D. a desorption drying stage: heating the mixture from-20 to-10 ℃ to 20 to 30 ℃ for 2 to 3 hours, and drying the mixture at the temperature of 20 to 30 ℃ for 3 to 4 hours;
E. and filling inert gas into the mixture after the desorption drying stage to obtain the interleukin 6 freeze-dried calibrator.
As a preferred embodiment of the method of the present invention, the degree of vacuum in step C is less than 30 Pa; and in the step D, the vacuum degree is less than 15 Pa.
In the technical scheme of the invention, the method for preparing the interleukin 6 freeze-drying calibrator comprises the stages of pre-freezing, sublimation drying and desorption drying, improves the thermal stability of the interleukin 6 antigen, protects the activity of the interleukin 6 antigen to the greatest extent, is favorable for the transportation of the freeze-dried interleukin 6 antigen, and prolongs the storage time of the interleukin 6 calibrator and the interleukin 6 detection kit.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a freeze-drying protective solution which can maintain the shape of a freeze-dried interleukin 6 calibrator, is not sticky to a bottle, does not collapse, and has good toughness and good re-solubility;
(2) the invention provides a freeze-drying protective solution, which can keep the activity of an interleukin 6 calibrator before and after freeze-drying basically unchanged, can obviously improve the stability of the interleukin 6 calibrator, is beneficial to the transportation of freeze-drying antigen, prolongs the storage time of the interleukin 6 calibrator and prolongs the storage time of an interleukin 6 detection kit;
(3) the invention provides a method for preparing interleukin 6 freeze-dried calibrator, which improves the thermal stability of interleukin 6 antigen after vacuum freeze-drying, protects the activity of IL-6 antigen to the maximum extent and prolongs the storage time.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1A Freeze-drying protective solution
A freeze-drying protective solution comprises the following components in percentage by weight:
trehalose 0.5%, gamma-cyclodextrin 1%, mannitol 1%, collagen protective agent 0.5%, tween-80 0.1% and KY100 0.1%, the balance being Tris-HCL buffer solution, the concentration of Tris-HCL buffer solution being 20 mM; wherein the pH value of the freeze-drying protective solution is 7.4.
The preparation method of the collagen protective agent comprises the following steps:
s1, extracting collagen by an enzymolysis method: cleaning fresh Corii Sus Domestica, removing pig hair, removing skin with scalpel, cutting into pieces of about 1cm × 1cm, cleaning with phosphate of pH7.5, soaking and washing with purified water for 5 times, adding 100mL of purified water, heating at 60 deg.C for 2 hr, taking out, cutting, weighing 5g into a beaker, adding 10% NaCl, soaking and stirring for 1h, degreasing, filtering to obtain filter residue, adding acetic acid with the mass concentration of 0.5mol/L, pepsin (the unit of enzyme activity is 2000U/mL) and compound protease (the unit of enzyme activity is 2000U/mL), stirring and soaking for 12h, controlling pH within 2-3, performing enzymolysis and centrifugation, centrifuging for 10min at a speed of 5000r/min, separating and taking supernatant, adding 50% polyethylene glycol 6000 into the supernatant to salt out collagen, centrifuging for 15min (at a speed of 8000r/min), and collecting precipitate to obtain collagen;
s2, purifying collagen: and (3) adding 10mL of distilled water into the collagen prepared in the step S1, dissolving in a conical flask, placing in a water bath kettle at 37 ℃ for starting reaction, heating in a boiling water bath for 5min for inactivating enzyme after the reaction is finished, centrifuging to obtain a supernatant, centrifuging for 15min at the centrifugation speed of 10000r/min, purifying the supernatant by using a cross-linked dextran G25 desalting column, and obtaining the collagen protective agent as the short peptide with the molecular weight cutoff of less than 5 kd.
A preparation method of a freeze-drying protective solution comprises the step of mixing Tris buffer solution, trehalose, gamma-cyclodextrin, mannitol, a collagen protective agent, Tween-80 and KY100 according to the mass concentration to prepare the freeze-drying protective solution.
An interleukin 6 freeze-drying calibrator is prepared by mixing an interleukin 6 calibrator with the freeze-drying protective solution in the embodiment.
Example 2A Freeze-drying protective solution
A freeze-drying protective solution comprises the following components in percentage by weight:
trehalose 0.5%, gamma-cyclodextrin 0.5%, mannitol 0.5%, collagen protectant 0.2%, tween-80 0.05% and KY100 0.1%; the balance is Tris-HCL buffer solution, and the concentration of the Tris-HCL buffer solution is 20 mM; wherein the pH value of the freeze-drying protective solution is 7.4.
In this example, the collagen protectant was prepared in the same manner as in example 1.
An interleukin 6 freeze-drying calibrator is prepared by mixing an interleukin 6 calibrator with the freeze-drying protective solution in the embodiment.
Example 3A Freeze-drying protective solution
A freeze-drying protective solution comprises the following components in percentage by weight:
trehalose 0.5%, gamma-cyclodextrin 0.5%, mannitol 0.5%, collagen protectant 0.2%, tween-80 0.05% and KY100 0.1%; the balance is HEPES-Na buffer solution with the concentration of 10 mM; wherein the pH value of the freeze-drying protective solution is 7.4.
In this example, the collagen protectant was prepared in the same manner as in example 1.
An interleukin 6 freeze-drying calibrator is prepared by mixing an interleukin 6 calibrator with the freeze-drying protective solution in the embodiment.
Example 4A Freeze-drying protective solution
A freeze-drying protective solution comprises the following components in percentage by weight:
1% trehalose, 2% gamma-cyclodextrin, 2% mannitol, 0.5% collagen protectant, 0.1% tween-80 and 0.1% KY 100; the balance is Tris-HCL buffer solution, and the concentration of the Tris-HCL buffer solution is 20 mM; wherein the pH value of the freeze-drying protective solution is 7.2.
In this example, the collagen protectant was prepared in the same manner as in example 1.
An interleukin 6 freeze-drying calibrator is prepared by mixing an interleukin 6 calibrator with the freeze-drying protective solution in the embodiment.
Example 5A Freeze-drying protective solution
A freeze-drying protective solution comprises the following components in percentage by weight:
50mM Tris-HCL buffer solution, 3% trehalose, 5% gamma-cyclodextrin, 5% mannitol, 1% collagen protective agent, 0.1% Tween-80 and 0.1% KY 100; the balance is Tris-HCL buffer solution, and the concentration of the Tris-HCL buffer solution is 50 mM; wherein the pH value of the freeze-drying protective solution is 7.6.
In this example, the collagen protectant was prepared in the same manner as in example 1.
An interleukin 6 freeze-drying calibrator is prepared by mixing an interleukin 6 calibrator with the freeze-drying protective solution in the embodiment.
Example 6A method for preparing an interleukin 6 lyophilized calibrator by using a lyophilized protection solution
A method for preparing an interleukin 6 freeze-drying calibrator by using the freeze-drying protective solution comprises the following steps:
A. mixing: mixing the interleukin 6 calibrator with the freeze-drying protective solution to obtain a mixture;
B. a pre-freezing stage: c, placing the mixture prepared in the step A in a vacuum freeze dryer to be frozen for 2 hours at the temperature of minus 30 ℃;
C. a sublimation drying stage: heating from-30 ℃ to-15 ℃ for 1h, drying the mixture subjected to the pre-freezing stage at-15 ℃ for 12h, wherein the vacuum degree is less than 30 Pa;
D. a desorption drying stage: heating from-15 deg.C to 25 deg.C for 2h, drying at 25 deg.C for 4h, and keeping vacuum degree below 15 Pa;
E. and (4) filling nitrogen into the mixture after the desorption drying stage to obtain the interleukin 6 freeze-dried calibrator.
Example 7 method for preparing interleukin 6 lyophilized calibrator by using lyophilized protection solution
A method for preparing an interleukin 6 freeze-drying calibrator by using the freeze-drying protective solution comprises the following steps:
A. mixing: mixing the interleukin 6 calibrator with the freeze-drying protective solution to obtain a mixture;
B. a pre-freezing stage: c, placing the mixture prepared in the step A in a vacuum freeze dryer to be frozen for 3 hours at the temperature of minus 30 ℃;
C. a sublimation drying stage: heating from-30 ℃ to-15 ℃ for 1h, drying the mixture subjected to the pre-freezing stage at-15 ℃ for 10h, wherein the vacuum degree is less than 30 Pa;
D. a desorption drying stage: heating from-15 deg.C to 25 deg.C for 2h, drying at 25 deg.C for 4h, and keeping vacuum degree below 15 Pa;
E. and (4) filling nitrogen into the mixture after the desorption drying stage to obtain the interleukin 6 freeze-dried calibrator.
Comparative example 1A Freeze-drying protective solution
Similar to example 1, except that no collagen protectant was included, the remaining raw materials were the same as in example 1.
Comparative example 2A Freeze-drying protective solution
Similar to example 1, except that trehalose was not included, the remaining raw materials were the same as in example 1.
Comparative example 3A Freeze-drying protective solution
Similar to example 1, except that mannitol was not included, the remaining starting material was the same as in example 1.
Comparative example 4A Freeze-drying protective solution
Similar to example 1, except that no gamma-cyclodextrin was included, the remaining starting material was the same as in example 1.
Test example I, evaluation of physical Properties
The interleukin 6 calibrator is mixed with the freeze-drying protection solutions prepared in the examples 1-5 and the comparative examples 1-4 respectively to prepare the interleukin 6 freeze-drying calibrator by the preparation method of the example 7, then the interleukin 6 freeze-drying calibrator obtained by the respective preparation methods is respectively placed in the environments of 2-8 ℃ and 25 ℃ for storage, a control group is set, 20mM Tris-HCl buffer solution and the interleukin 6 calibrator are mixed to prepare the interleukin 6 calibrator of the control group, and freeze-drying is carried out according to the same freeze-drying process. Wherein the freeze-drying protection solutions prepared in examples 1-5 and comparative examples 1-4 were tested at different concentrations of 0.5ng/mL and 0.05ng/mL, respectively, and the properties and residual water content of the interleukin 6 freeze-drying calibrator and the interleukin 6 calibrator of the control group were regularly observed, as shown in tables 1 and 2.
TABLE 1
And (4) conclusion: the results in table 1 show that the interleukin 6 calibrator prepared in the control group is easy to agglomerate after being freeze-dried, and the interleukin 6 calibrator agglomerates are respectively placed in the environment of 2-8 ℃ and 25 ℃ and adhered to the wall of the bottle; compared with a control group, the interleukin 6 calibration product prepared in the embodiments 1-5 of the invention has the advantages of no shrinkage after freeze-drying, uniform properties after freeze-drying, no sticking to the bottle wall, good toughness, no sticking to a bottle plug, no agglomeration after the bottle plug is stored for 12 months at the temperature of 2-8 ℃ or 8 months at the temperature of 25 ℃, no sticking to the bottle, good toughness, no sticking to the bottle plug, stable physical properties, and the best physical properties of the embodiment 1.
In comparison with examples, comparative examples 1 to 4 lack one of the collagen protectant, trehalose, mannitol and γ -cyclodextrin, the physical properties were slightly inferior to those of examples 1 to 5, and the control group had no external shape and adhered to the inner wall of the lyophilization flask.
TABLE 2
And (4) conclusion: the interleukin 6 freeze-dried calibrator prepared in examples 1 to 5 has low residual moisture, and the change of the residual moisture after long-term storage is small, which is beneficial to long-term storage.
Test example two evaluation of resolubility
The interleukin 6 freeze-drying calibrator obtained by using the freeze-drying protection solutions of the examples 1-5 and the comparative examples 1-4 is used as an experimental group, 20mM Tris-HCl buffer solution and the interleukin 6 calibrator are mixed to be used as a control group, purified water is added into the experimental group and the control group respectively and then the experimental group and the control group are kept still for 10s, the dissolution condition of the interleukin 6 freeze-drying calibrator is observed, and the result shows that the interleukin 6 freeze-drying calibrator of the experimental group can be completely dissolved, the solution is clear, and no obvious block or foam is generated; the interleukin 6 freeze-dried calibrator of the comparative example group has the phenomena of difficult dissolution and turbidity and poor re-solubility; the interleukin 6 freeze-dried calibrator of the control group has slow dissolution speed, and partial floccule can be still seen after standing for 10min, so that the re-solubility is poor.
Test example III evaluation of Activity
The interleukin 6 lyophilized calibrator obtained by using the lyophilized protection solutions of examples 1 to 5 and comparative examples 1 to 4 was used as an experimental group, and a 20mM Tris-HCl buffer solution was mixed with the interleukin 6 calibrator as a control group, and the activities of each group before and after lyophilization were measured, and the results are shown in Table 3.
TABLE 3
The data in table 3 show that the activity of the interleukin 6 lyophilized calibrator in the control group is obviously reduced, compared with the control group, the activity of the interleukin 6 calibrator prepared in examples 1-5 before and after lyophilization is basically unchanged, and the activity difference between the interleukin 6 calibrator prepared in comparative examples 1-4 before and after lyophilization is within 5%, and the activity difference between the interleukin 6 calibrator prepared in comparative examples 1-4 before and after lyophilization is more than 10%, which indicates that the activity of the interleukin 6 calibrator before and after lyophilization can be effectively maintained by compounding various components including collagen protectant, trehalose, mannitol and gamma-cyclodextrin.
Test example four evaluation of stability
The interleukin 6 freeze-drying calibrator obtained by using the freeze-drying protective solution of the examples 1-5 and the comparative examples 1-4 is used as an experimental group, 20mM Tris-HCl buffer solution and the interleukin 6 calibrator are mixed to be used as a control group, and the interleukin 6 calibrator of the experimental group and the control group is placed at 37 ℃ for 10 days and is placed at 4 ℃ for 30 days after being opened to detect the activity. The results are shown in Table 4.
TABLE 4
The results in table 4 show that the interleukin 6 freeze-dried calibrators of examples 1 to 5 have poor stability compared with the control group; the interleukin 6 calibrator can keep good activity and has good stability under different experimental groups. The freeze-drying protective solution provided by the invention can be used for remarkably improving the stability of the interleukin 6 calibrator, facilitating the transportation of freeze-dried antigen, prolonging the storage time of the interleukin 6 calibrator and prolonging the storage time of the interleukin 6 detection kit.
In conclusion, the interleukin 6 freeze-drying calibrator prepared by the freeze-drying protective solution disclosed by the invention is free from shrinkage and collapse; the freeze-drying shape is uniform and stable; the bottle is not stuck; the toughness is better; the bottle stopper does not stick to the interleukin 6 freeze-drying calibrator; and the solution is kept still for 10s after adding purified water, the interleukin 6 freeze-dried calibrator can be completely dissolved, and the solution is clear without block and foam; the activity of the interleukin 6 calibrator is basically kept unchanged before and after freeze-drying; the stability of the interleukin 6 calibrator can be obviously improved, the transportation of freeze-dried antigen is facilitated, the storage time of the interleukin 6 calibrator is prolonged, and the storage time of the interleukin 6 detection kit is prolonged.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (5)
1. A freeze-drying protective solution for interleukin 6 calibrator is characterized by comprising the following components in percentage by weight: 0.5-3% of trehalose, 0.5-5% of gamma-cyclodextrin, 0.5-5% of mannitol, 0.2-1% of collagen protective agent, 0.05-0.1% of tween 80, 0.05-0.1% of KY100 and the balance of Tris-HCl buffer solution or HEPES-Na buffer solution, wherein the concentration of the Tris-HCl buffer solution or the HEPES-Na buffer solution is 10mM-50 mM; the pH value of the freeze-drying protective solution is 7.2-7.6;
the preparation method of the collagen protective agent comprises the following steps:
s1, extracting collagen by an enzymolysis method: cleaning animal skin, adding purified water, heating, taking out, cutting, degreasing, filtering to obtain filter residue, adding acetic acid, pepsin and compound protease, stirring, soaking, performing enzymolysis, centrifuging to obtain supernatant, adding polyethylene glycol 6000, performing collagen salting-out, centrifuging to obtain precipitate to obtain collagen;
s2, purifying collagen: heating the collagen prepared in the step S1 in a boiling water bath to inactivate enzymes, centrifuging to obtain a supernatant, purifying the supernatant, and intercepting short peptides with molecular weight less than 5kd to obtain a collagen protective agent; the enzyme activity of the pepsin and the compound protease is 2000U/mL.
2. The lyoprotectant of claim 1, comprising, in weight percent: 0.5% of trehalose, 1% of gamma-cyclodextrin, 1% of mannitol, 0.5% of collagen protective agent, 0.1% of surfactant, 0.1% of preservative and the balance of Tris-HCl buffer solution, wherein the concentration of the Tris-HCl buffer solution is 20 mM.
3. An interleukin 6 lyophilized calibrator obtained by mixing an interleukin 6 calibrator with the lyophilized protection solution according to any one of claims 1 to 2.
4. A method for preparing a lyophilized calibrator for interleukin 6 using the lyophilized protection solution according to any one of claims 1 to 2, comprising the steps of:
A. mixing: mixing an interleukin 6 calibrator with the lyoprotectant according to any of claims 1-2 to form a mixture;
B. a pre-freezing stage: c, freezing the mixture prepared in the step A for 2-3 hours at the temperature of-35 to-25 ℃;
C. a sublimation drying stage: raising the temperature from-35 to-25 ℃ to-20 to-10 ℃, wherein the temperature raising time is 0.5 to 1 hour, and drying the mixture subjected to the pre-freezing stage for 10 to 12 hours at the temperature of-20 to-10 ℃;
D. a desorption drying stage: heating the mixture from-20 to-10 ℃ to 20 to 30 ℃ for 2 to 3 hours, and drying the mixture at the temperature of 20 to 30 ℃ for 3 to 4 hours;
E. and filling inert gas into the mixture after the desorption drying stage to obtain the interleukin 6 freeze-dried calibrator.
5. The method of claim 4, wherein the vacuum in step C is less than 30 Pa; and in the step D, the vacuum degree is less than 15 Pa.
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