A buffer for reconstituting the freeze-dried IL-1β antigen

Abstract

The present invention provides a buffer for reconstituting the lyophilized IL-1β antigen. It is discovered for the first time that Krovin 100 can enhance the reconstitution stability of the lyophilized IL-1β antigen. The reconstituted solution can be stored at 2-8°C for at least 7 days and at -20°C and -80°C for at least 15 days. On the one hand, the improvement of the stability of the IL-1β antigen protein is beneficial to improving the accuracy of the detection reagent. On the other hand, after the lyophilized product is reconstituted, the detection personnel can use it at any time instead of preparing it once for each detection. While facilitating the operation of the detection personnel, it is beneficial to reduce the manual operation error; combining a basic buffer, salts, inert proteins and Krovin 100 to prepare a buffer for reconstituting the lyophilized IL-1β antigen, and optimizing the pH, concentration, etc. to achieve the best effect; all components of the buffer are common components, and chemical reagents such as surfactants used in common reconstitution buffers are not used. The buffer is non-toxic and has a low reagent cost, and has great application potential.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic assays, and particularly to a buffer for reconstituting freeze-dried IL-1β antigen. Background Art

[0002] Interleukin-1 belongs to macrophage cytokines (Monokine) with a molecular weight of about 17 kD. The term IL-1 refers to two cytokines, IL-1α and IL-1β. In vivo, IL-1 is mainly responsible for the acute phase response, including fever, acute protein synthesis, anorexia, and lethargy. Through these mechanisms, cytokines of the IL-1 family become an important part of the host's defense against infection. IL-1β can promote the recruitment of inflammatory cells at the inflammation site by inducing the expression of endothelial cell adhesion molecules and the release of chemokines by stromal cells; it can also induce the production of various enzymes, mediate the release of inflammatory mediators prostaglandin E2 and NO, and mediate local and systemic inflammatory responses. The basis of many autoinflammatory diseases is an increased release of active IL-1β. For example, in patients with osteoarthritis, IL-1β stimulates the production of a variety of matrix metalloproteinases, leading to the rupture of connective tissue and inhibiting the production of collagen, thus having a negative impact on articular cartilage; at the same time, IL-1β has a direct and indirect stimulatory effect on the maturation of osteoclasts, mediating the development of bone erosion in arthritis. Therefore, the in vitro diagnostic assay of IL-1β has high clinical application value.

[0003] For in vitro immunoassay reagents, quality control products and calibration products are essential, and freeze-dried IL-1β antigen is essential for in vitro diagnostic assays. However, after the freeze-dried IL-1β antigen is reconstituted, its stability is extremely vulnerable to environmental factors, such as temperature, pH, oxidation, mechanical force, freeze-thaw, radiation, chemicals, microorganisms, etc. The components in the reconstitution buffer and the protective agents contained in the freeze-dried product may also affect the stability of the IL-1β antigen protein. After the antigen beads reconstituted with the existing IL-1β antigen freeze-dried product reconstitution buffer are placed for a period of time, they basically show unstable properties, low signal-to-noise ratio, jump value phenomenon, etc., and cannot protect the antigen well, which has a certain impact on sample assignment; during the period from the reconstitution of the freeze-dried product to the on-machine detection, the content of IL-1β antigen protein in it is very likely to change significantly, resulting in a large deviation in the detection results.

[0004] Therefore, there is an urgent need to propose a new buffer for the reconstitution of freeze-dried IL-1β antigen, so as to improve the stability of IL-1β antigen, reduce the signal-to-noise ratio, etc., to meet the requirements of in vitro diagnostic assays. Summary of the Invention

[0005] To solve the above problems, the present invention discloses a buffer for reconstituting the freeze-dried IL-1β antigen. The main components include bis-tris buffer, salts, inert proteins, and preservatives. After reconstituting the freeze-dried IL-1β antigen with this buffer, it can be used for subsequent immune platform-related tests, and has the advantages of good stability and high signal-to-noise ratio.

[0006] On the one hand, the present invention provides a buffer for reconstituting the freeze-dried IL-1β antigen, and the buffer includes Krovin 100.

[0007] Krovin 100 can improve the stability of the reconstitution of the freeze-dried IL-1β antigen. The reconstituted solution can make the reconstituted IL-1β antigen have high stability at 2-8°C and lower temperatures, and can be refrigerated and stored at 2-8°C and lower temperatures for at least 7 days.

[0008] Krovin 100 is a colorless, slightly viscous, oily liquid with a slightly aromatic odor. It can be miscible with water and polar organic solvents such as ethanol, propanol, and propylene glycol. It has excellent solubility and can be used to solubilize and dissolve various raw materials and active ingredients. Krovin 100 is usually used as a preservative due to its antimicrobial activity function. It can inhibit or even eliminate a large number of Gram-positive bacteria and negative microorganisms, and can also significantly inhibit a variety of common bacteria such as Escherichia coli E.coil and Staphylococcus aureus.

[0009] The present invention has discovered and demonstrated that Krovin 100 has the effect of improving the stability of the freeze-dried IL-1β antigen. On the one hand, it is due to its antibacterial effect. However, compared with other common preservatives, Krovin 100 has a better stability improvement effect, indicating that its effect does not entirely come from its antibacterial property. Krovin 100 does not provide obvious stability for the reconstitution of other protein freeze-dried products, indicating that its effect is likely related to the physical and chemical properties of Krovin 100 and the IL-1β antigen protein. Therefore, on the other hand, Krovin 100 may have functions such as protecting the three-dimensional structure of the antigen protein, maintaining the ionization state of the antigen protein, reducing the adsorption and aggregation of the reconstituted antigen protein, and reducing the electrostatic interaction of the antigen protein, thereby significantly improving the stability of the reconstituted freeze-dried IL-1β antigen, and also having the effect of inhibiting the interference of the reconstituted solution and improving the signal-to-noise ratio, which has never been found in the prior art.

[0010] In some embodiments, the pH of the Krovin 100 is 6.0-7.0.

[0011] Furthermore, the buffer further includes a basic buffer, and the pH of the basic buffer is 5.8-7.2.

[0012] In some embodiments, the pH of the base buffer is 6.0 - 6.8.

[0013] The base buffer is any solution with buffering function, which can stabilize the pH. The concentration of the base buffer determines the pH value of the buffer. The components and pH in the base buffer have a certain impact on the stability of the reconstitution of the freeze-dried antigen. Appropriate components and pH can reduce intermolecular interactions and maintain the ionization state of the antigen protein, thereby improving the stability after reconstitution. Krovin 100 has the effect of improving the stability when reconstituting IL-1β, and the best effect is achieved at pH 6.0 - 6.8, but it is not limited to this pH range, and the buffering effect can enhance its stability in the base buffer.

[0014] In some embodiments, the base buffer is bis-tris buffer: the pH value ranges from 5.8 to 7.2.

[0015] Preferably, the pH of the base buffer is 6.8. Under the condition of pH 6.8, the base buffer has a stronger stabilizing effect on the reconstitution of the IL-1β freeze-dried antigen. The reasons may be: on the one hand, when the pH is stabilized at 6.8, the decomposition rate of the IL-1β freeze-dried antigen is directly reduced, thereby improving its stability; on the other hand, Krovin 100 has a better effect of improving stability at pH 6.8.

[0016] Furthermore, the base buffer includes at least one of MOPS buffer, PBS buffer, HEPES buffer, MES buffer, Tris hydrochloride buffer, citrate buffer, bis-tris buffer.

[0017] Preferably, the base buffer includes at least one of MOPS buffer, PBS buffer, HEPES buffer, MES buffer, Tris hydrochloride buffer, citrate buffer, bis-tris buffer.

[0018] Furthermore, the base buffer includes 20 - 80 mmol / L bis-tris.

[0019] Preferably, the base buffer includes 50 mmol / L bis-tris.

[0020] Furthermore, the buffer further includes salts, and the salts include any one or more of NaCl and KCl.

[0021] Furthermore, the buffer includes 0.1% - 0.9% NaCl and 0.05% - 0.2% KCl.

[0022] Preferably, the buffer solution comprises 0.9% NaCl and 0.1% KCl.

[0023] Furthermore, the buffer solution further comprises an inert protein, and the inert protein comprises at least one of human serum albumin, bovine serum albumin, and casein.

[0024] Furthermore, the buffer solution comprises 5‰ - 20‰ bovine serum albumin.

[0025] In some ways, the effects of buffer solutions with different components on improving the stability of IL-1β freeze-dried product antigen after reconstitution were further studied. The experimental results showed that when the buffer solution system was changed or the concentrations of its various components were changed, the stability after reconstitution would be significantly reduced. Therefore, it can be known that there is a close synergistic relationship between Krovin 100 and specific buffer solution components and concentrations in terms of improving the stability of IL-1β freeze-dried product antigen after reconstitution. The synergistic effect formed by the optimal formulation in the present invention is the key to achieving good stability. Once the buffer solution system or the concentrations of its various components are changed, this synergistic effect is broken, and the stability after reconstitution will be significantly reduced. This further emphasizes that in the present invention, it is necessary to precisely control the components and concentrations of the buffer solution to ensure the best stabilizing effect in combination with Krovin 100, providing reliable conditional guarantees for the reconstitution and application of IL-1β freeze-dried product antigen.

[0026] On the other hand, the present invention provides a method for improving the reconstitution stability of IL-1β freeze-dried product antigen, which is to mix the buffer solution according to any one of the above technical solutions with the IL-1β freeze-dried product antigen.

[0027] On yet another hand, the present invention provides a product for detecting IL-1β, comprising the buffer solution for reconstituting the IL-1β freeze-dried product antigen according to any one of the above technical solutions.

[0028] The product can be any product for detecting IL-1β protein, and the product form can be a test strip, a kit, an analyzer, etc.

[0029] For example, in the form of a kit product, the kit can be a kit for detecting IL-1β. The IL-1β freeze-dried product antigen is used as a quality control product or a calibration product, and the buffer solution of the present invention can be used as a protective solution during the freeze-drying process of IL-1β.

[0030] The purchased antigen freeze-dried product is dissolved according to the dissolution method recommended by the manufacturer, generally dissolved with purified water. The antigen concentration is higher than the actual concentration used, and it needs to be diluted with a diluent. If it is not freeze-dried, the stability is poor and it is not conducive to product preservation. In order to enable the product to be stored for a long time, the antigen is freeze-dried. After freeze-drying, the antigen can be stored for 24 months. In this process, the formula of the diluent is screened. The buffer provided by the present invention can be used as the diluent during the freeze-drying process of IL-1β, and also as the protective liquid during the freeze-drying process. The buffer provided by the present invention can improve the stability of the calibrator or control product, which is beneficial to improving the accuracy and sensitivity of detection.

[0031] Freezing and thawing will affect the stability of the product. The calibrator reconstituted with this buffer can be frozen and thawed at least 4 times.

[0032] On the other hand, the present invention provides a use of Krovin 100 for preparing a reagent for improving the antigen reconstitution stability of IL-1β freeze-dried products.

[0033] The beneficial effects of the present invention include:

[0034] 1. It is first discovered that Krovin 100 can improve the antigen reconstitution stability of IL-1β freeze-dried products. The reconstituted solution can be refrigerated and stored at 2-8°C and lower temperatures for at least 7 days. On the one hand, the improvement of the stability of IL-1β antigen protein is beneficial to the improvement of detection accuracy. On the other hand, the detection personnel can take the reconstituted solution at any time instead of preparing it once for each detection. While facilitating the operation of the detection personnel, it is beneficial to reduce manual operation errors;

[0035] 2. Combine a basic buffer, salts, inert proteins, and Krovin 100 to prepare a buffer for reconstituting the antigen of IL-1β freeze-dried products, and optimize the pH, concentration, etc. to achieve the best effect;

[0036] 3. All components of the buffer are common components, and chemical reagents such as surfactants in common reconstitution solutions are avoided. The buffer is non-toxic and the reagent cost is low, with great application potential.

[0037] 4. After the antigen of the IL-1β freeze-dried product reconstituted with this buffer is frozen and thawed 4 times, the antigen stability is good. Specific Embodiments

[0038] The preferred embodiments of the present invention will be further described in detail below. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not limit it in any way. All the features disclosed in the embodiments of the present invention, or all the steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.

[0039] Example 1: Preparation of buffer for reconstituting freeze-dried IL-1β antigen and reconstitution of freeze-dried product

[0040] Prepare according to the concentration: 50 mM bis-tris, 0.9% NaCl, 0.1% KCl, 10‰ BSA, 2‰ Krovin 100 (Sigma-Aldrich, catalog number: ACN0054A), pH 6.8. Obtain the buffer for reconstituting freeze-dried IL-1β antigen.

[0041] Add 1 mL of the above purified water to 10 μg of IL-1β freeze-dried antigen (Thermo Fisher, catalog number: PMC0815), let it stand for 20 - 30 min, and invert and mix 10 times. Dilute the antigen with the above buffer for reconstituting freeze-dried IL-1β antigen to 6 concentrations: 0 pg / mL, 8 pg / mL, 40 pg / mL, 200 pg / mL, 1000 pg / mL, and 5000 pg / mL. Freeze-dry the above 6 concentrations of antigen into spherical products with a diameter of 2.5 - 2.8 mm using a liquid nitrogen freeze-drying device, specifically, from low to high IL-1β concentration: small ball No. 0, small ball No. 1, small ball No. 2, small ball No. 3, small ball No. 4, and small ball No. 5; after reconstituting the 6 freeze-dried small balls with the above buffer for reconstituting freeze-dried IL-1β antigen, the concentrations are 0 pg / mL, 8 pg / mL, 40 pg / mL, 200 pg / mL, 1000 pg / mL, and 5000 pg / mL respectively.

[0042] I. Stability experiment

[0043] Set up multiple experimental groups for reconstitution experiments on small balls No. 0 - 5. After reconstitution, one group is directly tested on the machine, and the other groups are stored at room temperature for 3 h, 6 h, 9 h; stored at 2 - 8°C for 1 day, 3 days, 7 days, 10 days; stored at -20°C for 1 day, 3 days, 7 days, 10 days, 15 days, 20 days; stored at -80°C for 1 day, 3 days, 7 days, 10 days, 15 days, 20 days, and then tested on the machine. The instrument reads and records the RLU value (luminescence intensity), compares the test value with the value directly detected after reconstitution, calculates the deviation, deviation = measured luminescence value after low-temperature storage / initial value of luminescence value directly detected after reconstitution - 1. The results are shown in Table 1 - Table 4, and Table 5 shows the comparison results of the reconstitution buffer of the present invention with other commonly used buffers (the other commonly used buffers are specifically described in Comparative Document 1 and Comparative Document 2, Comparative Document 1 is a Chinese patent application with the application publication number CN118091118A, and Comparative Document 2 is a Chinese invention patent with the authorization announcement number CN117368493B).

[0044] Table 1: Deviation caused by storage at room temperature after reconstitution

[0045]

[0046] Table 2: Deviations caused by storage at 2 - 8°C after reconstitution

[0047]

[0048] Table 3: Deviations caused by storage at - 20°C after reconstitution

[0049]

[0050] Table 4: Deviations caused by storage at - 80°C after reconstitution

[0051]

[0052] Table 5: Comparison of the stability of antigen beads reconstituted with different reconstitution buffers (at 2 - 8°C, 10 days)

[0053]

[0054] 2 - 8°C is a commonly used low - temperature storage temperature. At this temperature, the reconstitution buffer of the present invention can make the antigen titers of all beads have a deviation lower than or close to 10% within 10 days of storage. Therefore, the reconstitution buffer of the present invention plays a better role in maintaining the stability of antigen beads with different concentrations.

[0055] II. Sensitivity experiment

[0056] In addition to improving the reconstitution stability of freeze - dried products, the reconstitution buffer of the present invention can also improve the detection sensitivity of the kit. In the cytokine kit, the lyophilized IL - 1β antigen is used as a calibrator. Respectively, the solvent of the interleukin recombinant protein solution in Calibration Product 1 in the above - mentioned Comparative Document and the sample diluent in Comparative Document 2, and the buffer for reconstituting the lyophilized IL - 1β antigen of the present invention are used as calibrator buffers. A double - antibody sandwich method is used to detect IL - 1β with a commercially available antibody. The IL - 1β blank sample is repeatedly detected 20 times, and the average value M and standard deviation SD value of the measurement results are calculated. The limit of blank is M + 2SD, and the results are shown in Table 6.

[0057] Table 6: Limits of blank of IL - 1β with different calibrator buffers (pg / mL)

[0058]

[0059] It can be seen that the reconstitution buffer of the present invention, as a calibrator buffer, can improve the detection sensitivity. The reconstitution buffer of the present invention can increase the stability of the calibrator protein during the processes of reconstitution, preparation of the calibration solution, and detection, thus ensuring the stability of the detection results and improving the sensitivity.

[0060] III. Influence on the number of freeze - thaw cycles

[0061] Freeze-thaw means storing the antigen solution at low temperature, freezing it and then dissolving it again, which is considered as one freeze-thaw process; the freeze-thaw operation is repeated, and the number of cycles is the number of freeze-thaw times; the stability of the antigen during the freeze-thaw cycle can reflect the temperature changes that may be encountered during transportation or laboratory operations, and the number of freeze-thaw times the antigen remains stable can reflect the effect of the reconstitution buffer on the stability of the antigen lyophilized product in actual scenarios.

[0062] The solvent of the calibrator interleukin recombinant protein solution in the above-mentioned comparative document 1 (Chinese invention patent application with application publication number CN118091118A) and the sample diluent in comparative document 2 (Chinese invention patent with authorization announcement number CN117368493B) and the reconstitution buffer in Example 1 were used to reconstitute the No. 1 freeze-dried pellet, and the reconstituted solution was subjected to freeze-thaw experiments at -20°C and -80°C. After each freeze-thaw, samples were taken and the RLU value of the antigen was detected. Compared with the RLU value of the original reconstituted antigen pellet, the deviation was calculated, and the results are shown in Table 7.

[0063] Table 7: Freeze-thaw cycles and stability

[0064]

[0065] According to the above experimental results, the buffer in the patent cannot protect the antigen pellet during the freeze-thaw cycle. At -20°C, the deviation after the second freeze-thaw has exceeded 15%, indicating that some antigen proteins are denatured or degraded during the freeze-thaw process;

[0066] The reconstitution buffer in Example 1 subjected the solution of the reconstituted lyophilized pellet No. 1 to a freeze-thaw cycle. After 4 freeze-thaw cycles at -20°C and -80°C, the antigen stability was good, indicating that the reconstitution buffer can also play a role in improving the stability during the freeze-thaw process of the antigen pellet, and can play a role in improving the stability in actual application scenarios.

[0067] 4. Stability of freeze-dried pellets

[0068] In the kit containing freeze-dried pellet antigen calibrators or quality control products, the reconstitution buffer of the present invention is used as a freeze-drying protection solution for the calibrators or quality control products. Table 8 shows the protection of the overall freeze-drying process when the solvent of the calibrator interleukin recombinant protein solution in comparative patent 1, the sample diluent in comparative patent 2, and the reconstitution buffer in Example 1 are used as freeze-drying protection solutions for the calibrators and quality control products. After freeze-drying, the calibrators or quality control products are uniformly reconstituted with purified water for detection.

[0069] Table 8: Data of freeze-dried protective solution before and after freeze-drying (pg / mL)

[0070]

[0071] As can be seen from Table 8, when the calibration product uses the buffer solutions of Comparative Patent 1 and Comparative Patent 2 as the lyoprotectant, the detection results have a large deviation before and after lyophilization. Using the reconstitution buffer solution of Example 1 as the lyoprotectant results in a smaller deviation before and after lyophilization. It can be seen that the reconstitution buffer solution of the present invention can be used as a lyoprotectant to ensure the stability of the freeze-dried bead antigen calibration product or quality control product, thereby improving the accuracy of detection.

[0072] Example 2: Comparison of different reconstitution basic buffer solutions

[0073] When the research group of the present invention initially reconstituted the IL-1β lyophilized antigen, various reconstitution buffer solutions were tried, and it was found that the stability of most of the reconstituted products was very poor. Among them, the reconstitution buffer solution of Formula 7 (in Example 1) was significantly better than other buffer solutions. Some of the formulas are as follows: MOPS buffer solution (PH = 6.8), PBS buffer solution (PH = 6.8), HEPES buffer solution (PH = 7.5), Tris hydrochloride buffer solution (PH = 7.5), MES buffer solution (PH = 6.0), citrate buffer solution (PH = 5.5), bis-tris buffer solution (PH = 6.8).

[0074] Formula 1: 100 mmol / L MOPS, 0.9% NaCl, 0.1% KCl, 10‰ BSA, 2‰ Krovin 100 (mass ratio);

[0075] Formula 2: 2 mmol / L K 2 HPO 4 ·12H 2 O, 14 mmol / L NaH 2 PO 4 ·12H 2 O, 0.9% NaCl, 0.1% KCl, 10‰ BSA, 2‰ Krovin 100 (mass ratio);

[0076] Formula 3: 20 mmol / L HEPES, 0.9% NaCl, 0.1% KCl, 10‰ BSA, 2‰ Krovin 100 (mass ratio);

[0077] Formula 4: 50 mmol / L Tris hydrochloride, 0.9% NaCl, 0.1% KCl, 10‰ BSA, 2‰ Krovin 100 (mass ratio);

[0078] Formula 5: 100 mmol / L MES, 0.9% NaCl, 0.1% KCl, 10‰ BSA, 2‰ Krovin 100 (mass ratio);

[0079] Formulation 6: 10 mmol / L citric acid, 70 mmol / L sodium citrate, 0.9% NaCl, 0.1% KCl, 10‰ BSA, 2‰ Krovin 100 (by mass);

[0080] Formulation 7: 50 mmol / L bis-tris, 0.9% NaCl, 0.1% KCl, 10‰ BSA, 2‰ Krovin 100 (by mass) - the reconstitution buffer in Example 1;

[0081] The IL-1β freeze-dried antigen solutions reconstituted with reconstitution solutions of different formulations were stored in the following 4 storage environments (room temperature, 2 - 8°C, -20°C, and -80°C). Samples were taken at regular intervals, the RLU value (luminescence value) was measured, and the deviation was calculated compared with the initial value. The greater the absolute value of the deviation, the worse the stability of the IL-1β freeze-dried antigen solution.

[0082] I. Room Temperature

[0083] The above-mentioned reconstitution buffers were added to the IL-1β freeze-dried antigen, allowed to stand for 10 min, inverted and mixed 10 times, and the initial value was detected. Subsequently, it was placed at room temperature for 3 h, 6 h, and 9 h. The detected values and deviations during room temperature storage are shown in Tables 9 - 15.

[0084] Table 9: Formulation 1 at Room Temperature

[0085]

[0086] Table 10: Formulation 2 at Room Temperature

[0087]

[0088] Table 11: Formulation 3 at Room Temperature

[0089]

[0090] Table 12: Formulation 4 at Room Temperature

[0091]

[0092] Table 13: Formulation 5 at Room Temperature

[0093]

[0094] Table 14: Formulation 6 at Room Temperature

[0095]

[0096] Table 15: Formulation 7 at Room Temperature

[0097]

[0098] The experimental results show that the effects of Formulation 1 - Formulation 6 are poor, with a deviation of more than 10% after being placed at room temperature for 6 hours, indicating poor stability. Among them, Formulation 7 (the reconstitution buffer of Example 1) has the best effect, and even when placed at room temperature for 9 hours, the deviation can still be controlled within 10%.

[0099] II. 2 - 8°C

[0100] The reconstitution buffers of the above-mentioned Formulations 1 - 7 were respectively selected to reconstitute the IL-1β freeze-dried antigen. After shaking for 30 minutes to mix evenly, the initial value was detected, and it was placed at 2 - 8°C. The test values during placement are shown in Tables 16 - 22.

[0101] Table 16: Placement of Formulation 1 at 2 - 8°C

[0102]

[0103] Table 17: Placement of Formulation 2 at 2 - 8°C

[0104]

[0105] Table 18: Placement of Formulation 3 at 2 - 8°C

[0106]

[0107] Table 19: Placement of Formulation 4 at 2 - 8°C

[0108]

[0109] Table 20: Placement of Formulation 5 at 2 - 8°C

[0110]

[0111] Table 21: Placement of Formulation 6 at 2 - 8°C

[0112]

[0113] Table 22: Placement of Formulation 7 at 2 - 8°C

[0114]

[0115] The results show that when stored at the conventional 2 - 8°C, the decline in RLU value of Formulations 1 - 5 is more than 10% after 7 days of storage, indicating poor stability of the IL-1β freeze-dried antigen solution. The decline in RLU value of Formulation 6 is more than 15% after 10 days of storage, indicating poor stability of the IL-1β freeze-dried antigen. Only Formulation 7 can still control the decline in RLU value below 15% after 10 days of storage, and at some concentration gradients, the decline is below 10%. This shows that using Formulation 7 to reconstitute the IL-1β freeze-dried antigen can maintain stability.

[0116] III. Storage at -20°C

[0117] Using the reconstitution buffers of Formulas 1-7 in the same method as above, reconstitute the same batch of IL-1β freeze-dried antigen, detect the initial RLU value, and store it at -20°C for 1, 3, 7, 10, 15, and 20 days respectively. Detect the RLU value of the IL-1β freeze-dried antigen solution at the corresponding storage time and calculate the decrease rate compared to the initial value. The storage results of Formulas 1-7 are shown in Tables 23-29 respectively.

[0118] Table 23: Storage of Formula 1 at -20°C

[0119]

[0120] Table 24: Storage of Formula 2 at -20°C

[0121]

[0122] Table 25: Storage of Formula 3 at -20°C

[0123]

[0124] Table 26: Storage of Formula 4 at -20°C

[0125]

[0126] Table 27: Storage of Formula 5 at -20°C

[0127]

[0128] Table 28: Storage of Formula 6 at -20°C

[0129]

[0130] Table 29: Storage of Formula 7 at -20°C

[0131]

[0132] The results show that during storage at -20°C, although the low temperature provides a certain stabilizing effect, the decrease rates of the RLU values of Formulas 1-5 are all greater than 10% after 15 days of storage, indicating poor stability of the IL-1β freeze-dried antigen solution; the decrease rate of the RLU value of Formula 6 is greater than 15% after 20 days of storage, showing poor stability of the IL-1β freeze-dried antigen; only Formula 7 can control the decrease rate of the RLU value below 15% after 20 days of storage, and at some concentration gradients, the decrease rate is below 10%, indicating that reconstituting the IL-1β freeze-dried antigen with Formula 7 can maintain stability.

[0133] IV. Storage at -80°C

[0134] Using the same method as above, the same batch of IL-1β freeze-dried antigen was reconstituted with the reconstitution buffers of Formulas 1-7, the initial RLU value was measured, and it was stored at -80°C for 1, 3, 7, 10, 15, and 20 days respectively. The RLU values of the IL-1β freeze-dried antigen solutions corresponding to the storage times were measured and the decrease rate compared with the initial value was calculated. The storage results of Formulas 1-7 are shown in Tables 30-36 respectively.

[0135] Table 30: Formula 1 stored at -80°C

[0136]

[0137] Table 31: Formula 2 stored at -80°C

[0138]

[0139] Table 32: Formula 3 stored at -80°C

[0140]

[0141] Table 33: Formula 4 stored at -80°C

[0142]

[0143] Table 34: Formula 5 stored at -80°C

[0144]

[0145] Table 35: Formula 6 stored at -80°C

[0146]

[0147] Table 36: Formula 7 stored at -80°C

[0148]

[0149] The effects of Formulas 1-6 are less stable than those of the reconstitution buffer. Examining each group of experiments stored at -80°C, it was found that the deviation after 15 days of storage after reconstitution with Formulas 1-5 was slightly higher than 15%, and the RLU decrease rate after 20 days of storage after reconstitution with Formula 6 was greater than 15%. Even though low temperature provided a certain degree of stability, there was still obvious degradation of IL-1β. However, the reconstitution buffer of Formula 7 could ensure that the RLU decrease rate was within 10%, and the stability of the IL-1β freeze-dried antigen solution was the best.

[0150] Based on the above storage experiments at different temperatures, regardless of the storage temperature, the reconstitution buffer provided in Example 1 has the best stability after reconstitution.

[0151] Example 3: Effect of Krovin 100 on the reconstitution of the IL-1β freeze-dried antigen

[0152] In the study of the reconstitution process of the IL-1β freeze-dried antigen by the research group of the present invention, buffers of various components were tested, and Krovin 100 had particularly good effects. Since Krovin 100 is a preservative in the prior art and has antibacterial effects, comparative experiments were carried out with other similar preservatives, and the following were respectively prepared: 1. The optimal reconstitution buffer in Example 1; 2. bis-tris + 2‰ Krovin 100; 3. bis-tris + 2‰ Proclin 300; 4. bis-tris + 2‰ NaN 3 ; 5. bis-tris. After the No. 1 small ball was reconstituted, it was placed at 2-8°C for 1 day and 3 days respectively. Referring to the method in Example 2, the initial value, the measured value and the deviation were detected, and the deviation is shown in Table 37.

[0153] Table 37: Effects of different preservatives on the reconstitution stability of the IL-1β freeze-dried antigen

[0154]

[0155] According to the above experimental results, on the one hand, it shows that adding a preservative to the reconstitution solvent has a certain effect of increasing stability. However, compared with other types of preservatives, Krovin 100 has a better function of maintaining stability, indicating that in addition to its antibacterial and preservative effects, Krovin 100 also has other effects of increasing stability. On the other hand, it shows that the optimal buffer adopted in the present invention plays a key role in improving the reconstitution stability. Although only adding Krovin 100 can significantly improve the reconstitution stability of the IL-1β freeze-dried antigen, there is still a gap compared with the optimal scheme of the present invention. In this example, other buffers were compared with the optimal buffer in Example 1, further highlighting the importance of Krovin 100 and the buffer, and also indicating the indispensability of the synergistic effect between the buffer and Krovin 100 of the present invention. Only the synergy of the two can achieve the optimal reconstitution stability effect.

[0156] Based on the above results, the research team of the present invention hypothesized that the effect of Krovin 100 on the reconstitution stability of the IL-1β freeze-dried antigen might be due to a special effect of Krovin 100 on the IL-1β antigen protein. Therefore, in this example, creatine kinase isoenzyme freeze-dried products with similar low stability were reconstituted with bis-tris and bis-tris containing Krovin 100 respectively, and compared with the reconstitution of the IL-1β freeze-dried antigen. After reconstitution, the first small ball was placed at 2-8°C for 1 day and 3 days respectively. Referring to the method of Example 2, the initial value, measured value and deviation were detected, and the deviation is shown in Table 38.

[0157] Table 38: Effect of Krovin 100 on the reconstitution stability of different freeze-dried products

[0158]

[0159] The similarities between the creatine kinase isoenzyme freeze-dried product and the IL-1β freeze-dried product are as follows: the stability of both is vulnerable to environmental factors such as temperature, pH, oxidation, mechanical force, freeze-thaw, radiation, chemicals, microorganisms, etc., and the stability after reconstitution is low; the experimental results show that Krovin 100 only has an antiseptic and antibacterial effect on the reconstitution stability of creatine kinase isoenzyme freeze-dried products with similar low stability, slightly improving the stability, and the overall stability has not been significantly improved. Therefore, it is considered that Krovin 100 should have a special effect on the IL-1β antigen protein, which may be to protect the three-dimensional structure of the antigen protein, maintain the ionization state of the antigen protein, reduce the adsorption and aggregation of the antigen protein after reconstitution, and reduce the electrostatic interaction of the antigen protein, etc. The specific mechanism remains to be further studied.

[0160] Example 4: Composition of the reconstitution buffer

[0161] I. Importance of pH in improving the stability of the IL-1β freeze-dried antigen after reconstitution

[0162] According to the content of Example 3, Krovin 100 not only has an antiseptic and antibacterial effect on the IL-1β antigen protein, but also has an additional effect of maintaining stability, and this effect may be related to the physical and chemical properties of Krovin 100 and the IL-1β antigen protein. To further improve the effect of Krovin 100, relevant reagents were added to the reconstitution solvent in this example to further increase the stability of the IL-1β freeze-dried antigen after reconstitution.

[0163] First, a basic buffer solution is added to it. The basic buffer solution can provide a stable pH environment. In this embodiment, different concentrations of the basic buffer solution are prepared to achieve the corresponding pH. Except for the basic buffer solution, other components are the same as in Example 1. According to the method of Example 2, the deviations of the No. 1 microsphere after reconstitution and stored at 2-8°C for 1 day and 7 days are detected. The results are shown in Table 39.

[0164] Table 39: Stability of the reconstituted product stored at 2-8°C for 1 day and 7 days at different pH values

[0165]

[0166] According to the experimental results, compared with the reconstitution solvent without the basic buffer solution, after adding different basic buffer solutions, the stability of the IL-1β freeze-dried antigen after reconstitution can be improved by providing a stable pH environment. The stability is relatively high in the pH range of 6.0-6.8, and the stability is the best when the pH is 6.8. At this time, the basic buffer solution is bis-tris buffer solution; when the pH is 6.5-7.9, the basic buffer solution can be MOPS buffer solution; when the pH is 5.8-8.0, the basic buffer solution can be PBS buffer solution; when the pH is 6.8-8.2, the basic buffer solution can be HEPES; when the pH is 7-9, the basic buffer solution can be Tris buffer solution; when the pH is 5.5-6.7, the basic buffer solution can be MES buffer solution; when the pH is lower than 3.0-6.6, the basic buffer solution is citrate buffer solution; when the pH is 5.8-7.2, the basic buffer solution can be bis-tris;

[0167] The reason for the above improvement in stability may be that, on the one hand, the stable pH directly benefits the stability of the IL-1β antigen protein and reduces its degradation rate; on the other hand, the stable pH can enable Krovin 100 to play a good effect, and Krovin 100 has the best effect on the stability of the IL-1β antigen protein at pH 6.8.

[0168] In addition to pH, salts can be added to the buffer solution to maintain the stable ionization state of the IL-1β antigen protein, and inert proteins can be added to protect the IL-1β antigen protein. The salts can be NaCl, KCl, etc., and the inert proteins can be BSA, human serum albumin, casein, etc.

[0169] II. The importance of buffer components in improving the stability of the IL-1β freeze-dried antigen after reconstitution

[0170] Meanwhile, in this embodiment, the effect of buffers with different components on improving the stability of the IL-1β freeze-dried product after antigen reconstitution was further studied. To further improve the effect of Krovin 100, different buffer components were added to the reconstituted buffer in this embodiment in order to screen out the buffer formulation that can most effectively enhance the stability of the IL-1β freeze-dried product after antigen reconstitution. The screening of different formulations is as follows:

[0171] Formulation 1 (the best formulation in Example 1): Bis-tris buffer with a pH of 6.8, 0.9% NaCl, 0.1% KCl, 10‰ BSA, 2‰ Krovin 100;

[0172] Formulation 2: Bis-tris buffer with a pH of 6.8, 0.9% NaCl, 0.1% KCl, 10‰ human serum albumin, 2‰ Krovin 100;

[0173] Formulation 3: Bis-tris buffer with a pH of 6.8, 0.9% NaCl, 0.1% KCl, 1‰ casein, 2‰ Krovin100;

[0174] Formulation 4: Bis-tris buffer with a pH of 6.8, 0.9% NaCl, 0.1% KCl, 10‰ BSA, 2‰ Krovin500;

[0175] Formulation 5: Bis-tris buffer with a pH of 6.8, 0.9% NaCl, 0.1% KCl, 10‰ BSA, 2‰ Proclin300;

[0176] Formulation 6: Bis-tris buffer with a pH of 6.8, 10‰ BSA, 2‰ Krovin 100;

[0177] The specific results are shown in Table 40:

[0178] Table 40: Stability of the No. 1 small ball after reconstitution at 2-8°C for 1 day and 7 days under different buffers

[0179]

[0180] From the above experimental results, it can be seen that on the basis of the presence of Krovin 100, the components of the buffer also play a crucial role in improving the stability of the IL-1β freeze-dried product after antigen reconstitution. Different buffer systems and the concentration differences of their respective components will all have a significant impact on the stability after reconstitution.

[0181] Formulation 1, as the optimal formulation in Example 1 of the present invention, exhibits relatively better stability, while the stabilities of other formulations are significantly reduced to varying degrees. This indicates that when the buffer system is changed or the concentrations of its components are changed, the stability after reconstitution will be significantly reduced. Therefore, it can be seen that there is a close synergistic relationship between Krovin 100 and specific buffer components and concentrations in terms of improving the stability of the IL-1β freeze-dried antigen after reconstitution. The synergistic effect formed by the optimal formulation (Formulation 1) in Example 1 is the key to achieving good stability. Once the buffer system or the concentrations of its components are changed and this synergistic effect is broken, the stability after reconstitution will be significantly reduced. This further emphasizes that in the present invention, it is necessary to precisely control the components and concentrations of the buffer to ensure the best stabilizing effect in combination with Krovin 100, providing reliable conditional guarantees for the reconstitution and application of the IL-1β freeze-dried antigen.

[0182] The above-described embodiments have elaborated on the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, or substitutions in a similar manner made within the scope of the principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A buffer for re-dissolving a freeze-dried IL-1β antigen, characterized in that: The buffer solution consists of 50 mM bis-tris, 0.9% NaCl, 0.1% KCl, 10‰ BSA, and 2‰ Krovin 100; the pH of the bis-tris buffer solution is 6.

8.

2. A method for improving the stability of IL-1β lyophilized antigen reconstitution, characterized in that: The buffer solution according to claim 1 is mixed with the lyophilized IL-1β antigen.

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