Buffer solution for redissolving IL-1beta freeze-dried product antigen

Abstract

The invention provides a buffer solution for redissolving an IL-1beta freeze-dried product antigen. The improvement effect of Krovin 100 on the redissolution stability of the IL-1beta freeze-dried product antigen is found for the first time, and the redissolved solution can be stored at 2-8 DEG C for at least 7 days and at-20 DEG C and-80 DEG C for at least 15 days, so that on one hand, the improvement of the stability of the IL-1beta antigen protein is beneficial to the improvement of the accuracy of a detection reagent, and on the other hand, the improvement of the redissolution stability of the IL-1beta freeze-dried product antigen is facilitated; on the other hand, after the freeze-dried product is redissolved, detection personnel can take the freeze-dried product at any time instead of preparing the freeze-dried product once after detection, so that manual operation errors are reduced while operation of the detection personnel is facilitated; a basic buffer solution, salt, inert protein and Krovin 100 are combined to prepare a buffer solution for redissolving an IL-1beta freeze-dried product antigen, and the pH value, the concentration and the like are optimized to achieve the optimal effect; all components of the buffer solution are common components, chemical reagents such as a surfactant used in a common redissolving buffer solution are not used, and the buffer solution is non-toxic and low in reagent cost and has huge application potential.

Description

Technical Field

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

[0002] Interleukin-1 belongs to the category of macrophage cytokines (Monokine) with a molecular weight of approximately 17kD. The term IL-1 refers to two cytokines, IL-1α and IL-1β. In vivo, IL-1 is primarily responsible for acute phase responses, including fever, acute protein synthesis, anorexia, and lethargy. Through these mechanisms, IL-1 family cytokines become an important component of host defense against infection. IL-1β can promote the recruitment of inflammatory cells at the site of inflammation by inducing the expression of endothelial cell adhesion molecules and the release of chemokines by stromal cells; it can also induce the production of a variety of 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 the increased release of active IL-1β. For example, in patients with osteoarthritis, IL-1β stimulates the production of a variety of metalloproteinases, leading to the breakdown of connective tissue and inhibiting the production of collagen, which has 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 test for IL-1β has a high clinical application value.

[0003] For in vitro immunodiagnostic reagents, quality control products and calibration products are indispensable, and IL-1β antigen lyophilized products are indispensable in in vitro diagnostic testing. However, after the IL-1β antigen lyophilized products are reconstituted, their stability is easily affected by 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 lyophilized products may also affect the stability of the IL-1β antigen protein. After the existing IL-1β antigen lyophilized products are reconstituted with the reconstitution buffer, the antigen pellets are basically unstable, with a low signal-to-noise ratio and a jump value phenomenon after being placed for a period of time. The antigen cannot be well protected and has a certain impact on the sample assignment; during the period from the reconstitution of the lyophilized products to the machine detection, the IL-1β antigen protein content is likely to change significantly, resulting in a large deviation in the test results.

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

[0005] To solve the above problems, the present invention discloses a buffer for re-dissolving lyophilized IL-1β antigen, the main components of which include bis-tris buffer, salts, inert protein and preservatives. After re-dissolving lyophilized 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] In one aspect, the present invention provides a buffer for reconstituting a freeze-dried IL-1β antigen, wherein the buffer comprises Krovin 100.

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

[0008] Krovin 100 is a colorless, slightly viscous, slightly aromatic oily liquid that is miscible with water and polar organic solvents such as ethanol, propanol, and propylene glycol. It has excellent solubility and can be used to solubilize a variety of raw materials and active ingredients. Krovin 100 is usually used as a preservative because of its antimicrobial activity. It can inhibit or even eliminate large doses of Gram-positive and Gram-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 discovers and demonstrates that Krovin 100 has the effect of improving the stability of IL-1β lyophilized antigen, which is due to its antibacterial effect on one hand. However, compared with other common preservatives, Krovin 100 has a better effect of improving stability, indicating that its effect does not all come from its antibacterial property. Krovin 100 does not provide obvious stability for the reconstitution of other protein lyophilized products, indicating that its effect is likely to be related to the physical and chemical properties of Krovin 100 and IL-1β antigen protein; therefore, on the other hand, Krovin 100 may have the functions of protecting the three-dimensional structure of the antigen protein, maintaining the ionization state of the antigen protein, reducing the adsorption and aggregation of the antigen protein after reconstitution, and reducing the electrostatic effect of the antigen protein, thereby significantly improving the stability of the IL-1β lyophilized antigen after reconstitution, and also has the effect of inhibiting the interference of the reconstitution solution, and improving the signal-to-noise ratio, which has never been found in the prior art.

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

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

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

[0013] Basal buffer is any solution with buffering function, which has the function of stabilizing pH. The concentration of basal buffer determines the pH value of the buffer. The components and pH in the basal buffer have a certain degree of influence on the stability of the reconstitution of lyophilized antigens. Appropriate components and pH can reduce the interaction between molecules and maintain the ionization state of antigen protein, thereby improving the stability after reconstitution. Krovin 100 has the function of improving the stability when reconstituted IL-1β, and the effect is best at pH 6.0-6.8, but it is not limited to this pH range. In the basal buffer, the buffering effect can improve its stability.

[0014] In some embodiments, the basic buffer is a bis-tris buffer with a pH value between 5.8 and 7.2.

[0015] Preferably, the pH of the basic buffer is 6.8. Under the condition of pH 6.8, the buffer has a stronger stabilizing effect on the reconstitution of the lyophilized IL-1β antigen. The reasons may be: on the one hand, when the pH is stabilized at 6.8, the decomposition rate of the lyophilized IL-1β 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 basic buffer comprises at least one of MOPS buffer, PBS buffer, HEPES buffer, MES buffer, Tris hydrochloric acid buffer, citric acid buffer, and bis-tris buffer.

[0017] Preferably, the basic buffer comprises at least one of MOPS buffer, PBS buffer, HEPES buffer, MES buffer, Tris hydrochloric acid buffer, citrate buffer and bis-tris buffer.

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

[0019] Preferably, the basic buffer comprises 50 mmol / L bis-tris.

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

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

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

[0023] Furthermore, the buffer 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 comprises 5‰ to 20‰ bovine serum albumin.

[0025] In some ways, the effect of buffers with different components on improving the stability of IL-1β lyophilized antigen after reconstitution was further studied. The experimental results show that when the buffer system or the concentration of each component therein is changed, the stability after reconstitution will be significantly reduced. Therefore, it can be seen that in terms of improving the stability of IL-1β lyophilized antigen after reconstitution, there is a close synergistic relationship between Krovin 100 and specific buffer components and concentrations. The synergistic effect formed by the optimal formula in the present invention is the key to achieving good stability. Once the buffer system or the concentration of each component therein is changed, this synergistic effect is broken, which will significantly reduce the stability after reconstitution. This also further emphasizes that in the present invention, it is necessary to accurately control the composition and concentration of the buffer to ensure the best stabilization effect with Krovin 100, and provide reliable conditions for the reconstitution and application of IL-1β lyophilized antigen.

[0026] On the other hand, the present invention provides a method for improving the reconstitution stability of IL-1β lyophilized antigen, comprising mixing the buffer described in any one of the above technical solutions with the IL-1β lyophilized antigen.

[0027] In yet another aspect, the present invention provides a product for detecting IL-1β, comprising the buffer for re-dissolving the lyophilized IL-1β antigen as described in any one of the above technical solutions.

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

[0029] For example, in the form of a kit, the kit can be a kit for detecting IL-1β, the lyophilized IL-1β antigen is used as a quality control product or a calibrator, and the buffer of the present invention can be used as a protective liquid during the lyophilization process of IL-1β.

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

[0031] Freeze-thaw cycles can affect product stability. Calibrators reconstituted with this buffer can be frozen and thawed at least 4 times.

[0032] In yet another aspect, the present invention provides a use of Krovin 100 for preparing a reagent for improving the reconstitution stability of IL-1β lyophilized antigen.

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

[0034] 1. For the first time, it was discovered that Krovin 100 can improve the stability of the reconstituted IL-1β lyophilized antigen. The reconstituted solution can be stored in a refrigerated environment at 2-8°C or lower for at least 7 days. On the one hand, the improvement of the stability of the IL-1β antigen protein is conducive to the improvement of detection accuracy. On the other hand, the testers can use the reconstituted solution at any time instead of preparing it once for each test, which is convenient for the testers to operate and helps to reduce manual operation errors.

[0035] 2. Combine basic buffer, salts, inert protein and Krovin 100 to prepare the buffer for re-dissolving IL-1β lyophilized antigen, and optimize pH, concentration, etc. to achieve the best effect;

[0036] 3. The various components of the buffer solution are all common components, and avoid the use of chemical reagents such as surfactants in common reconstitution solutions. The buffer solution is non-toxic, the reagent cost is low, and it has great application potential.

[0037] 4. After the IL-1β lyophilized antigen was reconstituted with this buffer and frozen and thawed 4 times, the antigen had good stability. DETAILED DESCRIPTION

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

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

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

[0041] Add 1 mL of the above purified water to 10 μg of IL-1β lyophilized antigen (Thermo Fisher Scientific, Cat. No.: PMC0815), let stand for 20-30 minutes, and mix by inversion 10 times. Dilute the lyophilized IL-1β antigen to 6 concentrations of 0 pg / mL, 8 pg / mL, 40 pg / mL, 200 pg / mL, 1000 pg / mL, and 5000 pg / mL using the buffer used to reconstitute the lyophilized IL-1β antigen. The above six concentrations of antigens were freeze-dried into spherical products with a diameter of 2.5-2.8 mm using a liquid nitrogen freeze-drying device. Specifically, they were No. 0 pellet, No. 1 pellet, No. 2 pellet, No. 3 pellet, No. 4 pellet and No. 5 pellet according to the IL-1β concentration from low to high; the concentrations of the six freeze-dried pellets after being reconstituted with the above-mentioned buffer for reconstituting the IL-1β freeze-dried antigen were 0 pg / mL, 8 pg / mL, 40 pg / mL, 200 pg / mL, 1000 pg / mL and 5000 pg / mL respectively.

[0042] 1. Stability test

[0043] Set up multiple experimental groups to conduct re-dissolution experiments on beads 0-5. After re-dissolution, one group was directly tested on the machine, and the other groups were stored at room temperature for 3h, 6h, and 9h; stored at 2-8℃ for 1 day, 3 days, 7 days, and 10 days; stored at -20℃ for 1 day, 3 days, 7 days, 10 days, 15 days, and 20 days; stored at -80℃ for 1 day, 3 days, 7 days, 10 days, 15 days, and 20 days, and then tested on the machine. The instrument read and recorded the RLU value (luminescence intensity) and compared the test value with the value directly detected after re-dissolution. , calculate the deviation, deviation = luminescence value tested after low-temperature storage / initial luminescence value directly detected after reconstitution - 1, the results are shown in Tables 1 to 4, Table 5 is the comparison results of the reconstitution buffer of the present invention and other commonly used buffers (the other commonly used buffers are specifically described in Comparative Documents 1 and 2, Comparative Document 1 is a Chinese invention patent application with application publication number CN118091118A, and Comparative Document 2 is a Chinese invention patent with authorization announcement number CN117368493B).

[0044] Table 1: Deviations 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 pellets reconstituted with different reconstitution buffers (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 titer of all beads have a deviation of less than 10% 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 of different concentrations.

[0055] 2. Sensitivity Experiment

[0056] In addition to improving the reconstitution stability of the lyophilized product, the reconstitution buffer of the present invention can also improve the detection sensitivity of the kit. In the cytokine kit, the IL-1β lyophilized product antigen is used as a calibrator, and the solvent of the calibrator interleukin recombinant protein solution in the above-mentioned comparative document 1 and the sample diluent in comparative document 2, and the buffer of the reconstituted IL-1β lyophilized product antigen of the present invention are used as the calibrator buffer, and the commercially available antibody is used to detect IL-1β by double antibody sandwich method, and the IL-1β blank sample is selected and repeatedly tested 20 times, and the mean value M and standard deviation SD value of the measurement results are calculated, and the blank limit is M+2SD. The results are shown in Table 6.

[0057] Table 6: IL-1β blank limits (pg / mL) for different calibrator buffers

[0058]

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

[0060] 3. Impact on freeze-thaw times

[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 buffer solutions of comparative patents 1 and 2 are used as freeze-drying protective solutions for the calibrators, the deviation of the test results before and after freeze-drying is large. When the reconstitution buffer solution of Example 1 is used as the freeze-drying protective solution, the deviation of the test results before and after freeze-drying is small. It can be seen that the reconstitution buffer solution of the present invention can be used as a freeze-drying protective solution to ensure the stability of freeze-dried pellet antigen calibrators or quality control products, thereby improving the accuracy of the test.

[0072] Example 2: Comparison of different reconstitution base buffers

[0073] The research team of the present invention initially tried a variety of reconstitution buffers when reconstituted the IL-1β freeze-dried antigen and found that most of them had poor stability after reconstitution. Among them, the reconstitution buffer of formula 7 (in Example 1) was significantly better than other buffers. Some of the formulas are as follows: MOPS buffer (PH=6.8), PBS buffer (PH=6.8), HEPES buffer (PH=7.5), Tris hydrochloric acid buffer (PH=7.5), MES buffer (PH=6.0), citric acid buffer (PH=5.5), and bis-tris buffer (PH=6.8).

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

[0075] Formula 2: 2mmol / LK2HPO4·12H2O, 14mmol / LNaH2PO4·12H2O, 0.9%NaCl, 0.1%KCl, 10‰BSA, 2‰Krovin 100 (mass ratio);

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

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

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

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

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

[0081] The IL-1β lyophilized antigen solution reconstituted with different formulations of reconstitution solution was stored in the following 4 storage environments (room temperature, 2-8℃, -20℃ and -80℃), taken out at regular intervals, and the RLU value (luminescence value) was measured. The deviation was calculated by comparing with the initial value. The larger the absolute value of the deviation, the worse the stability of the IL-1β lyophilized antigen solution.

[0082] 1. Room temperature

[0083] The above-mentioned reconstitution buffers were added to the IL-1β freeze-dried antigen, allowed to stand for 10 minutes, inverted and mixed 10 times, and the initial value was detected. Then, the mixture was placed at room temperature for 3 hours, 6 hours, and 9 hours. The detection values ​​and deviations at room temperature are shown in Tables 9 to 15.

[0084] Table 9: Formulation 1 Place at room temperature

[0085]

[0086] Table 10: Formulation 2 Place at room temperature

[0087]

[0088] Table 11: Formulation 3 placed at room temperature

[0089]

[0090] Table 12: Formulation 4 placed at room temperature

[0091]

[0092] Table 13: Formulation 5 placed at room temperature

[0093]

[0094] Table 14: Formulation 6 Place at room temperature

[0095]

[0096] Table 15: Formulation 7 Place at room temperature

[0097]

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

[0099] 2.2-8℃

[0100] The reconstitution buffers of the above formulas 1 to 7 were respectively selected to reconstitute the IL-1β freeze-dried antigen, and the mixture was shaken for 30 minutes to mix well. The initial value was detected and the mixture was placed at 2-8°C. The test values ​​are shown in Tables 16 to 22.

[0101] Table 16: Formulation 1 2-8℃ Storage

[0102]

[0103] Table 17: Formulation 2 2-8℃ Storage

[0104]

[0105] Table 18: Formulation 3 2-8℃ Storage

[0106]

[0107] Table 19: Formulation 4 2-8℃ Storage

[0108]

[0109] Table 20: Formulation 5 2-8℃ Storage

[0110]

[0111] Table 21: Formulation 6 2-8℃ Storage

[0112]

[0113] Table 22: Formulation 7 2-8℃ Storage

[0114]

[0115] The results showed that when stored at the conventional 2-8°C, the RLU values ​​of Formulations 1-5 decreased by more than 10% after 7 days of storage, indicating that the stability of the IL-1β lyophilized antigen solution was poor; the RLU value of Formulation 6 decreased by more than 15% after 10 days of storage, indicating that the stability of the IL-1β lyophilized antigen was poor; only Formulation 7 could control the RLU value decrease to less than 15% after 10 days of storage, and at some concentration gradients, the decrease was less than 10%, indicating that the use of Formulation 7 to reconstitute the IL-1β lyophilized antigen can maintain stability.

[0116] 3. Store at -20℃

[0117] The same method as above was used to reconstitute the same batch of IL-1β lyophilized antigen using the reconstitution buffer of Formula 1-7, and the initial RLU value was detected. The lyophilized antigen was stored at -20°C for 1, 3, 7, 10, 15, and 20 days, respectively. The RLU values ​​of the IL-1β lyophilized antigen solutions at the corresponding storage time were detected and the decrease compared with the initial value was calculated. The storage results of Formulas 1-7 are shown in Tables 23-29, respectively.

[0118] Table 23: Formulation 1 -20℃ storage

[0119]

[0120] Table 24: Formulation 2 -20℃ storage

[0121]

[0122] Table 25: Formulation 3 -20℃ storage

[0123]

[0124] Table 26: Formulation 4 -20℃ storage

[0125]

[0126] Table 27: Formulation 5 -20℃ storage

[0127]

[0128] Table 28: Formulation 6 -20℃ storage

[0129]

[0130] Table 29: Formulation 7 -20℃ storage

[0131]

[0132] The results showed that although the low temperature provided a certain stabilizing effect when stored at -20°C, the RLU values ​​of Formulas 1-5 decreased by more than 10% after 15 days of storage, indicating that the stability of the IL-1β freeze-dried antigen solution was poor; the RLU value of Formula 6 decreased by more than 15% after 20 days of storage, indicating that the stability of the IL-1β freeze-dried antigen was poor; only Formula 7 could control the RLU value decrease to less than 15% after 20 days of storage, and at the same time, the decrease was less than 10% in some concentration gradients, indicating that the use of Formula 7 to reconstitute the IL-1β freeze-dried antigen can maintain stability.

[0133] 4. Store at -80℃

[0134] The same method as above was used to reconstitute the same batch of IL-1β lyophilized antigen using the reconstitution buffer of Formula 1-7, and the initial RLU value was detected. The lyophilized antigen was stored at -80°C for 1, 3, 7, 10, 15, and 20 days, respectively. The RLU values ​​of the IL-1β lyophilized antigen solutions at the corresponding storage time were detected and the decrease compared with the initial value was calculated. The storage results of Formulas 1-7 are shown in Tables 30-36, respectively.

[0135] Table 30: Formulation 1 -80℃ storage

[0136]

[0137] Table 31: Formulation 2 -80℃ storage

[0138]

[0139] Table 32: Formulation 3 -80℃ storage

[0140]

[0141] Table 33: Formulation 4 -80℃ storage

[0142]

[0143] Table 34: Formulation 5 -80℃ storage

[0144]

[0145] Table 35: Formulation 6 -80℃ storage

[0146]

[0147] Table 36: Formulation 7 -80℃ storage

[0148]

[0149] The effects of formula 1 to formula 6 are less stable than the reconstitution buffer. After examining the experiments in each group stored at -80°C, it was found that the deviation of formula 1 to 5 was slightly higher than 15% after being reconstituted and stored for 15 days, and the RLU decrease of formula 6 was greater than 15% after being reconstituted and stored for 20 days. Even though low temperature provides a certain stability, IL-1β still has obvious degradation. The reconstitution buffer of formula 7 can ensure that the RLU decrease is within 10%, and the stability of IL-1β lyophilized antigen solution is the best.

[0150] Based on the above storage experiments at different temperatures, no matter at what temperature the storage is performed, the reconstitution buffer provided in Example 1 has the best stability for reconstitution.

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

[0152] In the study of the reconstitution process of IL-1β lyophilized antigen, the research team of the present invention tested buffer solutions with various components, and Krovin 100 had a particularly good effect. Since Krovin 100 is a preservative in the prior art and has an antibacterial effect, it was compared with other similar preservatives in the experiment, and the following were prepared: 1. The best reconstitution buffer in Example 1; 2. bis-tris + 2‰ Krovin 100; 3. bis-tris + 2‰ Proclin 300; 4. bis-tris + 2‰ NaN3; 5. bis-tris. After reconstitution, pellet No. 1 was placed at 2-8°C for 1 day and 3 days respectively, and the initial value, detection value and calculation deviation were detected in accordance with the method of Example 2. The deviation is shown in Table 37.

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

[0154]

[0155] According to the above experimental results, on the one hand, it is shown that adding preservatives to the reconstitution solvent has a certain effect of increasing stability, but Krovin 100 has a better function of maintaining stability than other types of preservatives, indicating that Krovin 100 has other effects of increasing stability in addition to the antibacterial and antiseptic effects; on the other hand, it is shown that the optimal buffer used 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 best solution of the present invention. In this example, other buffers are compared with the best buffer in Example 1, which further highlights the importance of Krovin 100 and the buffer, and also shows the indispensability of the synergistic effect of the buffer of the present invention and Krovin 100. The two work together to achieve the optimal reconstitution stability effect.

[0156] Based on the above results, the research team of the present invention speculated that the re-dissolution stabilization effect of Krovin 100 on the IL-1β lyophilized antigen may be due to the special effect of Krovin 100 on the IL-1β antigen protein. Therefore, in this example, creatine kinase isoenzyme lyophilized products with similar low stability were re-dissolved with bis-tris and bis-tris containing Krovin 100, respectively, and compared with the re-dissolution of IL-1β lyophilized antigen. After re-dissolution, pellet No. 1 was placed at 2-8°C for 1 day and 3 days, respectively, and the initial value, detection value and calculation deviation were detected in accordance with the method of Example 2. The deviation is shown in Table 38.

[0157] Table 38: Effect of Krovin 100 on the stability of different lyophilized products after reconstitution

[0158]

[0159] The similarities between creatine kinase isoenzyme freeze-dried products and IL-1β freeze-dried products are that the stability of both products is easily affected by 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 a preservative 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 believed that Krovin100 should play a special role in 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 effect of the antigen protein. The specific mechanism needs further study.

[0160] Example 4: Composition of reconstitution buffer

[0161] 1. The importance of pH in improving the stability of IL-1β lyophilized 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 physicochemical properties of Krovin 100 and the IL-1β antigen protein. In order to further improve the effect of Krovin 100, this example adds relevant reagents to the reconstituted solvent to further increase the stability of the IL-1β freeze-dried antigen after reconstitution.

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

[0164] Table 39: Stability after reconstitution at different pH values ​​at 2-8°C for 1 day and 7 days

[0165]

[0166] According to the experimental results, compared with the reconstitution solvent without basic buffer, the addition of different basic buffers can improve the stability of IL-1β lyophilized antigen after reconstitution by providing a stable pH environment. The stability is higher in the pH range of 6.0-6.8, among which the stability is best at pH 6.8, and the basic buffer at this time is bis-tris buffer; when the pH is 6.5-7.9, the basic buffer can be MOPS buffer; when the pH is 5.8-8.0, the basic buffer can be PBS buffer; when the pH is 6.8-8.2, the basic buffer can be HEPES; when the pH is 7-9, the basic buffer can be Tris buffer; when the pH is 5.5-6.7, the basic buffer can be MES buffer; when the pH is lower than 3.0-6.6, the basic buffer is citric acid buffer; when the pH is 5.8-7.2, the basic buffer can be bis-tris;

[0167] The reason for the above-mentioned improved stability may be that, on the one hand, a stable pH is directly beneficial to the stability of the IL-1β antigen protein and reduces its degradation rate; on the other hand, a stable pH enables Krovin 100 to exert a good effect, and Krovin 100 has the best effect on stabilizing the IL-1β antigen protein at a pH of 6.8.

[0168] In addition to pH, salts can be added to the buffer to maintain the ionization state of the IL-1β antigen protein stable, 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] 2. Importance of buffer composition in improving the stability of IL-1β lyophilized antigen after reconstitution

[0170] At the same time, this example further studies the effect of different buffer components on improving the stability of IL-1β lyophilized antigen after reconstitution. In order to further improve the effect of Krovin 100, this example adds different buffer components to the reconstituted buffer in order to screen for the buffer formula that can most effectively improve the stability of IL-1β lyophilized antigen after reconstitution. The screening of different formulas is as follows:

[0171] Formula 1 (optimal formula in Example 1): bis-tris buffer with a pH of 6.8, 0.9% NaCl, 0.1% KCl, 10‰ BSA, 2‰ Krovin 100;

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

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

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

[0175] Formula 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 pellet No. 1 after reconstitution at 2-8°C for 1 day and 7 days in different buffers

[0179]

[0180] From the above experimental results, it can be seen that, based on the presence of Krovin 100, the composition of the buffer also plays a crucial role in improving the stability of the IL-1β lyophilized antigen after reconstitution. Different buffer systems and the concentration differences of each component in them will have a significant impact on the stability after reconstitution.

[0181] Formula 1, as the best formula in Example 1 of the present invention, shows relatively good stability, while the stability of other formulas is significantly reduced to varying degrees. This shows that when the buffer system is changed or the concentration of each component is changed, the stability after reconstitution will be significantly reduced. Therefore, it can be seen that in terms of improving the stability of IL-1β lyophilized antigen after reconstitution, there is a close synergistic relationship between Krovin 100 and specific buffer components and concentrations. The synergistic effect formed by the best formula (Formula 1) in Example 1 is the key to achieving good stability. Once the buffer system or the concentration of each component therein is changed, this synergistic effect is broken, which will significantly reduce the stability after reconstitution. This also further emphasizes that in the present invention, it is necessary to accurately control the composition and concentration of the buffer to ensure the best stabilization effect with Krovin 100, and provide reliable conditions for the reconstitution and application of IL-1β lyophilized antigen.

[0182] The embodiments described above provide a detailed description of 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 intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A buffer for reconstituted IL-1β freeze-dried antigen, characterized in that: The buffer included Krovin 100.

2. The buffer according to claim 1, characterized in that The buffer solution also includes a basic buffer solution, and the pH of the basic buffer solution is 5.8-7.

2.

3. The buffer according to claim 2, characterized in that The basic buffer comprises at least one of a MOPS buffer, a PBS buffer, a HEPES buffer, a MES buffer, a Tris hydrochloric acid buffer, a citric acid buffer, and a bis-tris buffer.

4. The buffer according to claim 3, characterized in that The basic buffer includes 20-80 mmol / L bis-tris.

5. The buffer according to claim 1, characterized in that The buffer solution also includes salts, and the salts include any one or more of NaCl and KCl.

6. The buffer according to claim 5, characterized in that The buffer comprises 0.1% to 0.9% NaCl and 0.05% to 0.2% KCl.

7. The buffer according to claim 1, characterized in that The buffer further comprises an inert protein, wherein the inert protein comprises at least one of human serum albumin, bovine serum albumin and casein.

8. The buffer according to claim 7, characterized in that The buffer comprises 5‰ to 20‰ bovine serum albumin.

9. A method for improving the stability of IL-1β lyophilized antigen reconstitution, characterized in that: Mix the buffer according to any one of claims 1 to 8 with the lyophilized IL-1β antigen.

10. A product for detecting IL-1β, characterized in that: A buffer for re-dissolving the lyophilized IL-1β antigen according to any one of claims 1 to 8.

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