A kind of interleukin-2 preservation solution and its related application

Abstract

The invention discloses an interleukin-2 preservation solution and related applications thereof, and relates to the biological field. A nonionic surfactant and an anionic surfactant are added to the preservation solution, wherein the nonionic surfactant comprises triethanolamine monostearate, and the anionic surfactant comprises disodium undecylenamide MEA sulfosuccinate. The nonionic surfactant can effectively reduce the surface tension of the solution and improve the solubility of the protein; the anionic surfactant can destroy the non-covalent bonds between proteins, depolymerize protein polymers, and enhance their dispersibility; the two can synergistically effectively inhibit protein aggregation, enhance protein dispersibility and solubility, and improve the stability of interleukin-2, so that the interleukin-2 can maintain good stability without freeze-drying, does not need to be redissolved during use, is easy to use, and has low cost, thereby improving the accuracy and reliability of interleukin-2 detection results.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to an interleukin-2 preservation solution and related applications thereof. Background Art

[0002] Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), has a molecular weight of 15 kDa and is encoded by a single gene located in the q26–28 region of chromosome 4. Its spatial conformation consists of four antiparallel α-helical bundles. IL-2 is the first interleukin to be discovered and cloned, and its corresponding receptor is also the first type I cytokine receptor to be cloned and its structure resolved. IL-2 is mainly produced by T cells (especially CD4 + IL-2 is synthesized by T cells after being stimulated by antigens or mitogens; B cells, NK cells and monocytes-macrophages can also produce IL-2. IL-2 can promote the proliferation of effector T cells and the production of cytokines, activate NK cells to maintain natural killing power, promote B cell proliferation, activate macrophages, etc. It is an important regulatory factor in the body's immune network. The detection of IL-2 has become an important indicator for evaluating the body's immune function and is widely used in the diagnosis of autoimmune diseases, infectious diseases, inflammatory diseases, etc.

[0003] Most of the IL-2 assay kits currently on the market use chemiluminescence methodology, in which the calibrators are important components and are all in the form of lyophilized products. The main reason is that the IL-2 antigen used as the preparation of the calibrator is extremely unstable in conventional buffer solutions, and must be prepared as a lyophilized product to meet stability requirements. Although lyophilized powder is relatively stable, it has the following problems: (1) The preparation process is complicated and the cost is high; (2) The molecular structure of the antigen is easily destroyed during the lyophilization process, affecting the activity; (3) It needs to be reconstituted before use, and human operating errors may cause the concentration after reconstitution to deviate from the target value, resulting in inaccurate clinical test results; (4) The reconstituted calibrator is not easy to preserve and has a short shelf life.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a storage solution for interleukin-2 and related applications thereof.

[0006] The inventor has found through research that the current liquid reagents containing interleukin-2, such as calibration products or quality control products, have poor stability problems, mainly because the antigens in the solution form polymers through non-chemical bond interactions. This aggregation process affects the structure of the protein, resulting in poor solubility and stability of the antigen, and even gradually loses its immune activity, thereby affecting the authenticity of the test results. The embodiment of the present invention adds nonionic surfactants and anionic surfactants to the storage solution of interleukin-2. The nonionic surfactant can effectively reduce the surface tension of the solution and improve the solubility of the protein; the anionic surfactant can destroy the non-covalent bonds between proteins, depolymerize protein polymers, and enhance their dispersibility; the two can synergistically effectively inhibit protein aggregation, enhance protein dispersibility and solubility, and improve the stability of the liquid reagent, so that interleukin-2 can maintain good stability without freeze-drying, and can be stably stored for about 7 days in an accelerated stability test at a temperature of 37°C. For different concentrations of interleukin-2 antigens, the retention rate of the detection signal is still above 90%, preferably above 92%, more preferably above 95%, and optimally even above 97%. It can be stably stored for about 12 months under normal storage conditions (2-8°C), providing the necessary prerequisite for meeting the accuracy standards of interleukin-2 detection.

[0007] On the one hand, an embodiment of the present invention provides a preservation solution for interleukin-2, which includes: a buffer solution, a nonionic surfactant and an anionic surfactant; wherein the nonionic surfactant includes triethanolamine monostearate; and the anionic surfactant includes disodium undecylenamide MEA sulfosuccinate.

[0008] The molecular formula of triethanolamine monostearate is C 24 H 49 NO4, CAS number is 10248-74-5.

[0009] In some embodiments, the volume fraction of the nonionic surfactant in the preservation solution is 0.05% to 1%, specifically, it can be any one of 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, or a range between any two of them.

[0010] The molecular formula of disodium undecylenamide MEA sulfosuccinate is C 17 H 27 NNa2O8S, CAS number is 26650-05-5.

[0011] In some embodiments, the volume fraction of the anionic surfactant in the preservation solution is 0.05% to 1%, specifically, it can be any one of 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, or a range between any two of them.

[0012] In some embodiments, the buffer comprises one or more of a HEPES buffer, a Tris buffer, a MES buffer, a MOPS buffer, a phosphate buffer, and an acetate buffer.

[0013] The buffer in the storage solution provided in the embodiment of the present invention is a base solution to provide a suitable pH value and an isotonic ion environment. There is no particular limitation on the type of buffer, and any conventional buffer that is compatible with the stored protein (IL-2) and does not change the properties of the protein can be used in the present invention.

[0014] In some embodiments, the pH of the buffer is 5.5 to 8, and the concentration is 10 to 200 mmol / L. Specifically, the pH can be any one of 5.5, 6, 6.5, 7, 7.5, 8, or a range between any two thereof. The concentration can be any one of 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 mmol / L, or a range between any two thereof.

[0015] In some embodiments, the preservation solution further comprises: any one or more of a protein stabilizer, a carbohydrate stabilizer and a preservative.

[0016] The present invention has no particular restrictions on the types and concentrations of protein stabilizers, sugar stabilizers and preservatives, which can be selected based on conventional technical knowledge or purpose of use.

[0017] In some embodiments, the mass volume fraction of the protein stabilizer in the preservation solution is 0.5% to 2.5%, specifically, it can be any one of 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, or a range between any two of them.

[0018] The "mass volume fraction" in this article is the same as the "mass volume percentage" or "w / v %", where mass volume percentage = (mass of solute / total volume of solution) × 100%. For example, "mass volume fraction is 10%" means that 10g of solute is dissolved in 100mL of solution, and the mass volume fraction of the solute is 10%.

[0019] In some embodiments, the protein stabilizer includes any one or more of casein, fish gelatin, bovine serum albumin and skimmed milk powder.

[0020] In some embodiments, the mass volume fraction of the carbohydrate stabilizer in the preservation solution is 0.5% to 2.5%, specifically, it can be any one of 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25% and 2.5%, or a range between any two of them.

[0021] In some embodiments, the carbohydrate stabilizer includes any one or more of sucrose, trehalose, glucose and dextran.

[0022] In some embodiments, the preservative includes any one or more of Proclin 300, MIT, Bro, IPBC or NaN3.

[0023] In some embodiments, the preservative is Proclin 300, and the volume percentage of Proclin 300 in the preservation solution is 0.1% to 0.5%, specifically any one of 0.1%, 0.2%, 0.3%, 0.4% and 0.5%, or a range between any two of them.

[0024] On the other hand, an embodiment of the present invention provides use of the preservation solution as described in any of the above embodiments in the preservation of interleukin-2.

[0025] On the other hand, an embodiment of the present invention provides a solution containing interleukin-2, which includes: interleukin-2 antigen and any of the above-mentioned preservation solutions.

[0026] In some embodiments, the concentration of interleukin-2 antigen in the interleukin-2-containing solution varies according to different detection methods. Generally, interleukin-2 in the range of 0 to 2500 pg / mL can be stably stored.

[0027] In some embodiments, the working concentration of the interleukin-2 antigen is 10 to 2500 pg / mL, specifically any one or more of 10, 50, 100, 200, 500, 1000, 1500, 2000, and 2500 pg / mL.

[0028] In some embodiments, the solution containing interleukin-2 is any one of a calibrator and a quality control product.

[0029] On the other hand, an embodiment of the present invention provides a kit, which includes any of the above-mentioned solutions containing interleukin-2.

[0030] In some embodiments, the kit is an interleukin-2 detection kit.

[0031] In addition, an embodiment of the present invention also provides the use of the solution containing interleukin-2 as described in any of the above embodiments or the kit as described in any of the above embodiments in detecting interleukin-2 for purposes other than disease diagnosis or treatment.

[0032] The present invention has the following beneficial effects:

[0033] The present invention develops a new interleukin-2 preservation solution, in which a nonionic surfactant and an anionic surfactant are added, wherein the nonionic surfactant includes triethanolamine monostearate, and the anionic surfactant includes disodium undecylenamide MEA sulfosuccinate. Among them, the nonionic surfactant can effectively reduce the surface tension of the solution and improve the solubility of the protein; the anionic surfactant can destroy the non-covalent bonds between proteins, depolymerize protein polymers, and enhance their dispersibility; the two can synergistically effectively inhibit protein aggregation, enhance protein dispersibility and solubility, and improve the stability of liquid interleukin-2, so that it can maintain good stability without freeze-drying, does not need to be redissolved during use, is easy to use, and has low cost, thereby improving the accuracy and reliability of interleukin-2 detection results. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0035] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0036] Examples 1 to 13 and Comparative Examples 1 to 7

[0037] 1. Preparation of Calibrators

[0038] 1.1. Preparation of basic buffer

[0039] Basic buffer 1: Weigh 5.8 g disodium hydrogen phosphate dodecahydrate (disodium hydrogen phosphate), 0.59 g sodium dihydrogen phosphate dihydrate (sodium dihydrogen phosphate), 15 g casein, 15 g sucrose, and 2.5 mL preservative (Proclin 300), dissolve them in 700 ml purified water, mix well, and adjust the volume to 1000 mL to obtain basic buffer 1 with a pH of 7.4.

[0040] Basic buffer 2: Weigh 6.06 g of tris(hydroxymethyl)aminomethane (TRIS), dissolve and mix with 700 ml of purified water, add hydrochloric acid to adjust the pH to 8.0, then add 5 g of casein, 25 g of sucrose, and 2.5 mL of preservative (Proclin300), dissolve and mix, and then adjust the volume to 1000 mL to obtain basic buffer 2 with a pH of 8.0.

[0041] Basic buffer 3: Weigh 10.86 g of MES sodium salt, dissolve and mix with 700 ml of purified water, add hydrochloric acid to adjust the pH to 5.0, then add 25 g of casein, 5 g of sucrose, and 2.5 mL of preservative (Proclin300), dissolve and mix, and then adjust the volume to 1000 mL to obtain basic buffer 3 with a pH of 5.5.

[0042] Basic buffer 4: weigh 5.8 g disodium hydrogen phosphate dodecahydrate (disodium hydrogen phosphate), 0.59 g sodium dihydrogen phosphate dihydrate (sodium dihydrogen phosphate), 15 g bovine serum albumin, 15 g sucrose, and 5 mL preservative (Proclin300), dissolve them in 700 ml purified water, mix well, and make up to 1000 ml to obtain basic buffer 4 with a pH of 7.4.

[0043] Basic buffer 5: Weigh 5.8 g disodium hydrogen phosphate dodecahydrate (disodium hydrogen phosphate), 0.59 g sodium dihydrogen phosphate dihydrate (sodium dihydrogen phosphate), 15 g casein, 15 g trehalose, and 1 mL preservative (Proclin300), dissolve them in 700 ml purified water, mix well, and adjust the volume to 1000 mL to obtain basic buffer 5 with a pH of 7.4.

[0044] 1.2. Preparation of Calibrator Dilution Solution

[0045] Prepare calibrator dilutions by adding surfactant to the base buffer as shown in Table 1.

[0046] Table 1 Calibrator dilutions (concentrations are in volume percentage)

[0047]

[0048]

[0049] 1.3 Preparation of Calibrators

[0050] The above 20 kinds of calibration dilution solutions were used to dilute interleukin-2 antigen to prepare three concentration gradient liquid calibration products S1 to S3 (10 pg / mL, 500 pg / mL, 1000 pg / mL).

[0051] Test Example 1: Performance testing of the calibrants of each embodiment and comparative example

[0052] As a calibrator for diagnostic reagents, the shelf life of the diagnostic reagents is generally set to one year, so the long-term stability at 2-8°C is examined. At the same time, considering that the ambient temperature of the reagents will not always be maintained at 2-8°C during transportation, a high-temperature accelerated stability experiment is set up.

[0053] High temperature accelerated stability test process: Take a set of calibration samples from each embodiment and each comparative example, place them at 37°C for 7 days, then take them out for signal value measurement, compare them with the signal values ​​of the corresponding calibration samples stored at 2-8°C, and calculate the signal retention rate.

[0054] Long-term stability testing process: The calibrants of the above-mentioned embodiments and comparative examples were stored at 2-8°C, and the signal values ​​were measured at the 0th month and the 12th month, and compared with the corresponding calibrants stored at the 0th month, and the signal retention rate was calculated.

[0055] Test results

[0056] The test results of each embodiment and each comparative example are shown in Table 2 and Table 3 below.

[0057] Table 2 High temperature accelerated stability results

[0058]

[0059]

[0060]

[0061] Table 3 Long-term stability results

[0062]

[0063]

[0064]

[0065] The results show that: through the signal value retention rates of Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3 in Table 2 and Table 3, it can be seen that the signal retention rates are higher than those of the groups without adding nonionic surfactants or anionic surfactants, indicating that nonionic surfactants or anionic surfactants can stabilize the conformation of proteins in solution, prevent protein aggregation, and improve the stability of calibration products. Secondly, after the two surfactants work together, the signal value retention rate is increased from 82.6% to 85.4% using a single component to 96.0% to 97.4%, which produces unexpected technical effects, indicating that the two can work together to effectively reduce the surface tension of the solution and improve the solubility of proteins; they can also destroy the non-covalent bonds between proteins, depolymerize protein polymers, and enhance their dispersibility, thereby greatly improving the stability of liquid calibration products.

[0066] From the signal value retention rates of Example 1, Comparative Examples 4 and 5 in Tables 2 and 3, it can be seen that the signal value retention rates of Span 20 (non-ionic surfactant) used in Comparative Example 4 and Tween 20 (non-ionic surfactant) used in Comparative Example 5 are between 85.5% and 87.8%, that is, the effect of using Span 20 or Tween 20 in conjunction with disodium undecylenamide MEA sulfosuccinate is not obvious compared with triethanolamine monostearate.

[0067] From the signal value retention rates of Example 1, Comparative Examples 6 and Comparative Examples 7 in Tables 2 and 3, it can be seen that the signal value retention rates of the fatty alcohol ammonium sulfate (anionic surfactant) used in Comparative Example 6 and the sodium dodecyl sulfate (anionic surfactant) used in Comparative Example 7 are between 84.1% and 86.9%, that is, the effect of using fatty alcohol ammonium sulfate or sodium dodecyl sulfate in conjunction with ethanolamine monostearate is not obvious compared with disodium undecylenamide MEA sulfosuccinate.

[0068] Based on Example 1, Comparative Example 4, Comparative Example 5, Comparative Example 6 and Comparative Example 7, it can be seen that only by replacing the anionic surfactant disodium undecylenamide MEA sulfosuccinate or the non-ionic surfactant triethanolamine monostearate with other surfactants of the same type, there is no obvious improvement on the stability of the liquid calibrant, and there is no specific synergistic effect.

[0069] Further, through the signal value retention rates of Example 1, Example 2, Example 3, Example 4 and Example 5 in Table 2 and Table 3, it can be seen that the use of triethanolamine monostearate in a concentration range of 0.1% to 0.5% in conjunction with disodium undecylenamide MEA sulfosuccinate can ensure a signal retention rate of more than 90%, while the signal value retention rates of too low or too high concentrations are all lower than 90%, with the optimal concentration being 0.3%.

[0070] It can be seen from the signal value retention rates of Example 1, Example 6, Example 7, Example 8 and Example 9 in Table 2 and Table 3 that the use of disodium undecylenamide MEA sulfosuccinate in a concentration range of 0.1% to 0.5% in conjunction with triethanolamine monostearate can ensure a signal retention rate of more than 90%, while the signal value retention rates of too low or too high concentrations are all lower than 90%, with the optimal concentration being 0.3%.

[0071] From the signal value retention rates of Example 1, Example 10 and Example 11 in Table 2 and Table 3, it can be seen that the signal retention rates under different buffer systems are all above 95% and the differences are not large, indicating that the interleukin-2 protein can be stably preserved under different buffer systems.

[0072] From the signal value retention rates of Example 1 and Example 12 in Table 2 and Table 3, it can be seen that the signal retention rates of different protein protective agents are all above 95% and the differences are not large, indicating that interleukin-2 protein can be stably preserved under different protein protective agents.

[0073] From the signal value retention rates of Example 1 and Example 13 in Table 2 and Table 3, it can be seen that the signal retention rates of different carbohydrate protective agents are all above 95% and the differences are not large, indicating that interleukin-2 protein can be stably preserved under different carbohydrate protective agents.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A storage solution for interleukin-2, characterized in that: It includes: Buffer, nonionic surfactant and anionic surfactant; wherein the nonionic surfactant includes triethanolamine monostearate; and the anionic surfactant includes disodium undecylenamide MEA sulfosuccinate.

2. The preservation solution according to claim 1, characterized in that The volume fraction of the nonionic surfactant in the preservation solution is 0.05% to 1%; Optionally, the volume fraction of the non-ionic surfactant in the preservation solution is 0.1% to 0.5%.

3. The preservation solution according to claim 1, characterized in that The volume fraction of the anionic surfactant in the preservation solution is 0.05% to 1%; Optionally, the volume fraction of the non-ionic surfactant in the preservation solution is 0.1% to 0.5%.

4. The preservation solution according to claim 1, characterized in that The buffer comprises one or more of HEPES buffer, Tris buffer, MES buffer, MOPS buffer, phosphate buffer and acetate buffer; Optionally, the pH of the buffer solution is 5.5 to 8, and the concentration is 10 to 200 mmol / L.

5. The preservation solution according to any one of claims 1 to 4, characterized in that The preservation solution further comprises: any one or more of a protein stabilizer, a carbohydrate stabilizer and a preservative; Optionally, the mass volume fraction of the protein stabilizer in the preservation solution is 0.5% to 2.5%; Optionally, the protein stabilizer includes: any one or more of casein, fish gelatin, bovine serum albumin and skimmed milk powder; Optionally, the mass volume fraction of the carbohydrate stabilizer in the preservation solution is 0.5% to 2.5%; Optionally, the carbohydrate stabilizer includes any one or more of sucrose, trehalose, glucose and dextran; Optionally, the preservative includes any one or more of Proclin 300, MIT, Bro, IPBC or NaN3; Optionally, the preservative is Proclin 300, and the volume percentage of Proclin 300 in the preservation solution is 0.1% to 0.5%.

6. Use of the preservation solution according to any one of claims 1 to 5 in the preservation of interleukin-2.

7. A solution containing interleukin-2, characterized in that It includes: An interleukin-2 antigen and the storage solution according to any one of claims 1 to 5.

8. The interleukin-2-containing solution according to claim 7, characterized in that The working concentration of the interleukin-2 antigen is 10 to 2500 pg / mL; Optionally, the solution is any one of a calibrator and a quality control product.

9. A kit, characterized in that: It comprises the solution of claim 7 or 8; Optionally, the kit is an interleukin-2 detection kit.

10. Use of the solution according to claim 7 or 8 or the kit according to claim 9 in detecting interleukin-2 for purposes other than disease diagnosis or treatment.

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