Method for improving stability of monoclonal antibody containing free sulfydryl and application

Abstract

The invention provides a method for improving the stability of a monoclonal antibody containing free sulfydryl, and belongs to the technical field of biological detection. Chloroacetamide and cysteine are used for preparing a reagent of the monoclonal antibody containing the free sulfydryl, and the stability of the monoclonal antibody containing the free sulfydryl is improved by modifying the sulfydryl on the monoclonal antibody containing the free sulfydryl through the chloroacetamide and the cysteine. The phenomenon that the free sulfydryl-containing monoclonal antibody is unstable in a heating environment is greatly solved. And under the action of the protective liquid, the thermal stability at 37 DEG C for 7 days is improved from the initial 30% to nearly 100%. According to the method provided by the invention, the use stability, the storage stability and the transportation stability of the monoclonal antibody containing the free sulfydryl are greatly improved, and the method relates to the use of the monoclonal antibody containing the free sulfydryl in various fields of biochemistry, immunochemistry, medical diagnosis, food safety and the like.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a method and application for improving the stability of monoclonal antibodies containing free thiol groups. Background Technology

[0002] Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone, targeting only a specific antigenic epitope. Large quantities of highly homogeneous antibodies can be obtained through culture or intraperitoneal inoculation in mice. These antibodies exhibit consistent structure, amino acid sequence, and specificity, and, provided there are no mutations during culture, antibodies secreted at different times maintain the same structure and function. This type of monoclonal antibody cannot be obtained by other methods. Due to their high specificity, high purity, and good homogeneity, monoclonal antibodies are widely used in techniques such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassay, immunohistochemistry, and flow cytometry.

[0003] Due to the differences in properties among monoclonal antibodies, some highly specific and active monoclonal antibodies suffer from poor stability and easy aggregation after development, rendering them unsuitable as effective detection reagents in kits. Improving the stability of monoclonal antibodies to maintain consistent biological activity under a wider range of environmental conditions is a pressing issue for researchers. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a method for improving the stability of monoclonal antibodies containing free thiol groups. This method involves using chloroacetamide and cysteine ​​as reagents to prepare monoclonal antibodies containing free thiol groups, thereby modifying the thiol groups on the monoclonal antibodies with chloroacetamide and cysteine ​​to improve their stability.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for improving the stability of monoclonal antibodies containing free thiol groups, wherein chloroacetamide and cysteine ​​are used to prepare a reagent containing free thiol monoclonal antibodies, and the reagent containing free thiol monoclonal antibodies comprises chloroacetamide, cysteine ​​and the monoclonal antibody containing free thiol groups.

[0006] Furthermore, in the reagent containing free thiol monoclonal antibody, the mass percentage of chloroacetamide is 0.005%~0.02%, and the mass percentage of cysteine ​​is 0.005%~0.02%.

[0007] Furthermore, the reagent containing the free thiol monoclonal antibody also includes a protective solution prepared from a buffer, bovine serum albumin, cyclodextrin, arginine, and Proclin 300.

[0008] Furthermore, the buffer solution includes one or more of phosphate buffer, HEPES buffer, and Tris buffer.

[0009] Furthermore, the buffer solution is a phosphate buffer solution with a concentration of 0.05 mol / L.

[0010] Further, by weight, the protective solution comprises PBS buffer, pH=7.4, 0.05 mol / L; bovine serum albumin 2-10 g / L; cyclodextrin 0.5-2 g / L; arginine 0.01 mol / L-0.02 mol / L; and Proclin 300 0.5-1 g / L.

[0011] Further, by weight, the protective solution comprises PBS buffer, pH=7.4, 0.05 mol / L; bovine serum albumin 2 g / L; cyclodextrin 2 g / L; arginine 0.01 mol / L; and Proclin 300 0.5 g / L.

[0012] Furthermore, the protective solution comprises PBS buffer, pH=7.4, 0.05 mol / L; bovine serum albumin 2 g / L; cyclodextrin 1 g / L; arginine 0.02 mol / L; and Proclin 300 0.5 g / L.

[0013] Furthermore, the protective solution comprises PBS buffer, pH=7.4, 0.05 mol / L; bovine serum albumin 5 g / L; cyclodextrin 1 g / L; arginine 0.01 mol / L; and Proclin 300 1 g / L.

[0014] Further, by weight, the protective solution comprises PBS buffer, pH=7.4, 0.05 mol / L; bovine serum albumin 10 g / L; cyclodextrin 0.5 g / L; arginine 0.01 mol / L; and Proclin 300 1 g / L.

[0015] This invention also provides the application of the above-described method in the preparation of diagnostic kits. For example, it can be used as a protective solution for antibody standards in antinuclear antibody detection kits, and as a protective solution for antibody standards in autoimmune liver disease diagnostic kits.

[0016] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention provides a method for improving the stability of monoclonal antibodies containing free thiol groups. Chloroacetamide and cysteine ​​are used simultaneously as reagents for preparing monoclonal antibodies containing free thiol groups. Chloroacetamide is an alkylating agent, and its active part is a chloroacetyl group, which has the ability to alkylate the thiol group (-SH) on cysteine ​​residues. This reaction generates a stable thioether bond, thereby blocking the reactivity of the thiol group. The thiol group of cysteine ​​can oxidize the thiol group to form a disulfide bond (-SS-), which is a key force in maintaining the tertiary and quaternary structures of proteins, thus blocking the reactivity of the thiol group. Furthermore, cysteine ​​can cleave the already formed aggregated disulfide bonds, allowing the protein that has aggregated due to the thiol group to reform into a monomeric state. The reaction of chloroacetamide and cysteine ​​is more selective, reacting only with thiol groups and usually not with amino groups, thus requiring less modification to the antibody structure and better preserving antibody activity. Modifying the thiol group on monoclonal antibodies containing free thiol groups with chloroacetamide and cysteine ​​improves the stability of these antibodies. This greatly solves the problem of instability of monoclonal antibodies containing free thiol groups under heating conditions.

[0017] (2) The method for improving the stability of monoclonal antibodies containing free thiol groups provided by the present invention uses the protective solution to place them at 37°C for 7 days, and the thermal stability is increased from the initial 30% to nearly 100%.

[0018] (3) This invention greatly improves the stability of the use, storage and transportation of monoclonal antibodies containing free thiol groups, and involves the use of monoclonal antibodies containing free thiol groups in various fields such as biochemistry, immunochemistry, medical diagnosis and food safety. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the present invention clearer, embodiments of the present invention are described in detail below, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to reference are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0020] The following ingredients were used in the embodiments of this invention: chloroacetamide (National Pharmaceutical Group Co., Ltd., catalog number: XW017907201); L-cysteine ​​(National Pharmaceutical Group Co., Ltd., catalog number: 62007234); monoclonal antibody (Wuhan Biobio Biotechnology Co., Ltd., catalog number: PB-GBM-MAB1); bovine serum albumin (National Pharmaceutical Group Co., Ltd., catalog number: C134730100); cyclodextrin (National Pharmaceutical Group Co., Ltd., catalog number: 69009360); arginine (National Pharmaceutical Group Co., Ltd., catalog number: 62004034); and Proclin 300 (Aladdin, catalog number: P406395-100ml).

[0021] Example 1 This embodiment provides the following process for modifying monoclonal antibodies containing free thiol groups with chloroacetamide and cysteine: Chloroacetamide (0.2 g) and cysteine ​​(0.2 g) were dissolved separately in phosphate buffer (pH 7.4) (10 mL), and 6 mL of 1 mg / mL monoclonal antibody was prepared using phosphate buffer. The light chain variable region sequence is shown in SEQ ID NO.1, specifically: SSELTQDPAVSVALGQTVCITCQGDSLRCNYASWYQQKPGQAPLCVIYGHKNRCSGIPDRFSGSCSGSTASLTISGARAECEADYYCHSRDISNNFYLFGPGTKLTVLG; the heavy chain variable region sequence is shown in SEQ ID NO.2, specifically: QVQLQQSAGGFQPGGSLRLSCAASGFPFFSYAMRWVRAAPGRGLAWVSDIESAGRNARYADSVKGRFTISRDNAKATVYLQMSALTAEDTAVYYCARAKFFAMDVWGQGTMVTVSS.

[0022] Add 1 ml of monoclonal antibody to each of the five small beakers, then add (2.5 μl, 5 μl, 7.5 μl, 10 μl, 15 μl) of prepared chloroacetamide and (15 μl, 10 μl, 7.5 μl, 5 μl, 2.5 μl) of prepared cysteine ​​in sequence, and react at 37 °C for 24 hours.

[0023] After the reaction was completed, the reaction solution was placed into dialysis bags with a diameter of 6.4 mm and a molecular weight cutoff of 12-14 kDa. Dialysis was performed at 2-8℃ for two days with phosphate buffer (pH 7.4), and the buffer was changed 3 times a day during the process.

[0024] After dialysis, carefully pour the solution from the dialysis bag into a 5ml volumetric flask and dilute to volume with phosphate buffer (pH 7.4) to a concentration of 0.2mg / ml.

[0025] Similarly, 1 ml of monoclonal antibody stock solution (1 mg / ml) was diluted to 5 ml with phosphate buffer (pH 7.4), resulting in a concentration of 0.2 mg / ml.

[0026] Comparative Example 1 The process for providing chloroacetamide-modified monoclonal antibodies containing free thiol groups in this comparative example is as follows: Dissolve 0.2 g of chloroacetamide in 10 mL of phosphate buffer (pH 7.4) and prepare 6 mL of 1 mg / mL monoclonal antibody with phosphate buffer.

[0027] Add 1 ml of monoclonal antibody to each of the five small beakers, and then add (2.5 μl, 5 μl, 7.5 μl, 10 μl, 15 μl) of prepared chloroacetamide in sequence. React at 37°C for 24 hours.

[0028] After the reaction was completed, the reaction solution was placed into dialysis bags and dialyzed at 2-8℃ for two days with phosphate buffer (pH 7.4), changing the buffer solution three times a day during the process.

[0029] After dialysis, carefully pour the solution from the dialysis bag into a 5ml volumetric flask and dilute to volume with phosphate buffer (pH 7.4) to a concentration of 0.2mg / ml.

[0030] Similarly, 1 ml of monoclonal antibody stock solution (1 mg / ml) was diluted to 5 ml with phosphate buffer (pH 7.4), resulting in a concentration of 0.2 mg / ml.

[0031] The reagents prepared in Example 1 and Comparative Example 1 were subjected to thermal stability experiments, and the specific procedures are as follows: Five different concentrations of chloroacetamide and cysteine-modified chloroacetamide + cysteine-monoclonal antibody and monoclonal antibody (0.2 mg / ml) were each aliquoted into six vials (100 μl / vial) and stored at 4℃, 37℃ for 12 h, 37℃ for 24 h, 37℃ for 72 h, and 37℃ for 168 h for stability testing. A total of six monoclonal antibodies were tested, with five storage conditions for each antibody, for a total of 30 conditions.

[0032] Five different concentrations of chloroacetamide-modified chloroacetamide-monoclonal antibodies and monoclonal antibodies (0.2 mg / ml) were each aliquoted into five vials (100 μl / vial) and stored at 4°C, 37°C for 12 h, 37°C for 24 h, 37°C for 72 h, and 37°C for 168 h for stability testing. A total of 25 conditions were set up for each of the five monoclonal antibodies.

[0033] Monoclonal antibodies from 55 different conditions, including chloroacetamide + cysteine-monoclonal antibody and chloroacetamide-monoclonal antibody, were diluted 100,000 times with PBS buffer. 100 μl of each antibody was added to an ELISA plate pre-coated with the corresponding detection protein. After incubation at 37°C for 1 hour, the plate was washed 3 times. Then, 100 μl of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research Inc., catalog number: 115-035-003) was added to each well. After incubation at 37°C for 1 hour, the plate was washed 3 times. Then, 100 μl of TMB substrate was added to each well, and the plate was incubated at 37°C for 5 minutes. Finally, 50 μl of stop solution was added to each well, and the OD value was measured at 450 nm using an ELISA reader.

[0034] The OD values ​​measured at 37℃ for 12h, 24h, 72h, and 168h were compared with the OD value at 4℃ as the activity retention values ​​for each monoclonal antibody.

[0035] The results are shown in Table 1. The monoclonal antibody with the addition of 10 μl chloroacetamide + 5 μl cysteine ​​solution exhibited the best stability. At 37℃ for 168 h, the chloroacetamide + cysteine ​​monoclonal antibody retained 89% of its activity, while the monoclonal antibody retained 30% of its activity.

[0036] Table 1.

[0037] Comparative Example 2 The process by which this comparative example provides a cysteine-modified monoclonal antibody containing a free thiol group is as follows: Dissolve 0.2 g of cysteine ​​in 10 mL of phosphate buffer (pH 7.4) and prepare 6 mL of 1 mg / mL monoclonal antibody with phosphate buffer.

[0038] Add 1 ml of monoclonal antibody to each of the five small beakers, and then add (2.5 μl, 5 μl, 7.5 μl, 10 μl, 15 μl) of prepared cysteine ​​in sequence. React at 37°C for 24 hours.

[0039] After the reaction was completed, the reaction solution was placed into dialysis bags and dialyzed at 2-8℃ for two days with phosphate buffer (pH 7.4), changing the buffer solution three times a day during the process.

[0040] After dialysis, carefully pour the solution from the dialysis bag into a 5ml volumetric flask and dilute to volume with phosphate buffer (pH 7.4) to a concentration of 0.2mg / ml.

[0041] Similarly, 1 ml of monoclonal antibody stock solution (1 mg / ml) was diluted to 5 ml with phosphate buffer (pH 7.4), resulting in a concentration of 0.2 mg / ml.

[0042] The reagents containing free thiol monoclonal antibodies prepared in Example 1 and Comparative Example 1 were subjected to thermal stability tests, and the specific procedures are as follows: Five different concentrations of chloroacetamide and cysteine-modified chloroacetamide + cysteine-monoclonal antibodies, as well as monoclonal antibodies (0.2 mg / ml), were each aliquoted into six vials (100 μl / vial) and stored at 4°C, 37°C for 12 h, 37°C for 24 h, 37°C for 72 h, and 37°C for 168 h for stability testing. A total of six monoclonal antibodies were tested, with five storage conditions for each antibody, resulting in a total of 30 storage conditions.

[0043] Five different concentrations of cysteine-modified cysteine-monoclonal antibodies and monoclonal antibodies (0.2 mg / ml) were each aliquoted into five vials (100 μl / vial) and stored at 4°C, 37°C for 12 h, 37°C for 24 h, 37°C for 72 h, and 37°C for 168 h for stability testing. A total of 25 conditions were tested for each of the five monoclonal antibodies.

[0044] Monoclonal antibodies under 55 different conditions, including chloroacetamide + cysteine-monoclonal antibody and cysteine-monoclonal antibody, were diluted 100,000 times with PBS buffer. 100 μl of each antibody was added to an ELISA plate pre-coated with the corresponding detection protein. After incubation at 37°C for 1 hour, the plate was washed 3 times. Then, 100 μl of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research Inc., catalog number: 115-035-003) was added to each well. After incubation at 37°C for 1 hour, the plate was washed 3 times. Then, 100 μl of TMB substrate was added to each well, and the plate was developed at 37°C for 5 minutes. Finally, 50 μl of stop solution was added to each well, and the OD value was measured at 450 nm using an ELISA reader.

[0045] The OD values ​​measured at 37℃ for 12h, 24h, 72h, and 168h were compared with the OD value at 4℃ as the activity retention values ​​for each monoclonal antibody.

[0046] The results are shown in Table 2. The monoclonal antibody with the addition of 10 μl chloroacetamide + 5 μl cysteine ​​solution showed the best stability. At 37℃ for 168 h, the chloroacetamide + cysteine ​​monoclonal antibody retained 91% of its activity, while the monoclonal antibody retained 33% of its activity.

[0047] Table 2.

[0048] Example 2 In this embodiment, the components of the protective solution 1 are as follows: PBS buffer, pH=7.4, 0.05 mol / L; bovine serum albumin 2 g / L; cyclodextrin 2 g / L; arginine 0.01 mol / L; Proclin 300 0.5 g / L.

[0049] The preparation method of the protective solution described in this embodiment is as follows: Prepare a 0.05 mol / L phosphate buffer solution with a pH of 7.4. Weigh or measure the corresponding components into the buffer solution and stir evenly to dissolve them one by one. Filter the solution through a 0.22-micron filter membrane, bottle it, and store it at 2-8℃.

[0050] Example 3 In this embodiment, the components of the protective solution 2 are as follows: PBS buffer, pH=7.4, 0.05 mol / L; bovine serum albumin 2 g / L; cyclodextrin 1 g / L; arginine 0.02 mol / L; Proclin 300 0.5 g / L.

[0051] The preparation method of the protective solution described in this embodiment is as follows: Prepare a 0.05 mol / L phosphate buffer solution with a pH of 7.4. Weigh or measure the corresponding components into the buffer solution and stir evenly to dissolve them one by one. Filter the solution through a 0.22-micron filter membrane, bottle it, and store it at 2-8℃.

[0052] Example 4 In this embodiment, the protective solution 3 components are in the following proportions: PBS buffer, pH=7.4, 0.05 mol / L; bovine serum albumin 5 g / L; cyclodextrin 1 g / L; arginine 0.01 mol / L; Proclin 300 1 g / L.

[0053] The preparation method of the protective solution described in this embodiment is as follows: Prepare a 0.05 mol / L phosphate buffer solution with a pH of 7.4. Weigh or measure the corresponding components into the buffer solution and stir evenly to dissolve them one by one. Filter the solution through a 0.22-micron filter membrane, bottle it, and store it at 2-8℃.

[0054] Example 5 In this embodiment, the protective solution has the following components in the following proportions: PBS buffer, pH=7.4, 0.05 mol / L; bovine serum albumin 10 g / L; cyclodextrin 0.5 g / L; arginine 0.01 mol / L; Proclin 300 1 g / L.

[0055] The preparation method of the protective solution described in this embodiment is as follows: Prepare a 0.05 mol / L phosphate buffer solution with a pH of 7.4. Weigh or measure the corresponding components into the buffer solution and stir evenly to dissolve them one by one. Filter the solution through a 0.22-micron filter membrane, bottle it, and store it at 2-8℃.

[0056] The stability of the protective solutions for the reagents containing free thiol monoclonal antibodies prepared in Example 1 was investigated. The free thiol monoclonal antibodies were diluted to 0.2 mg / ml using the four protective solutions prepared in Examples 2-5, and then further diluted to 0.2 mg / ml using PBS. Five aliquots of each of the five free thiol monoclonal antibodies were dispensed and stored at 4°C for 12 h, 37°C for 24 h, 37°C for 72 h, and 37°C for 168 h for stability testing. A total of five monoclonal antibodies (Wuhan Biobio Biotechnology Co., Ltd., catalog number: PB-GBM-MAB1) with different protective solutions were used, and each monoclonal antibody had five storage conditions, totaling 25 conditions.

[0057] Monoclonal antibodies for 25 different conditions were diluted 100,000 times with PBS buffer. 100 μl of each antibody was added to an ELISA plate pre-coated with the corresponding detection protein. After incubation at 37°C for 1 hour, the plate was washed 3 times. Then, 100 μl of HRP-labeled goat anti-mouse secondary antibody (Jackson ImmunoResearch Inc., catalog number: 115-035-003) was added to each well. After incubation at 37°C for 1 hour, the plate was washed 3 times. Then, 100 μl of TMB substrate was added to each well, and the plate was incubated at 37°C for 5 minutes. Finally, 50 μl of stop solution was added to each well, and the OD value was measured at 450 nm using an ELISA reader.

[0058] The OD values ​​measured at 37℃ for 12h, 24h, 72h, and 168h were compared with the OD value at 4℃ as the activity retention values ​​for each monoclonal antibody.

[0059] The results are shown in Table 3. The monoclonal antibody diluted 4 times with the protective solution showed the best stability. After 168 hours at 37°C, the activity of the protected monoclonal antibody was 54% retained; the activity of HRP diluted with PBS was 28% retained.

[0060] Table 3.

[0061] Example 6 The thermostability of chloroacetamide + cysteine-monoclonal antibody protected by protective solution was investigated.

[0062] The chloroacetamide + cysteine-monoclonal antibodies were diluted to 0.2 mg / ml using the four protective solutions prepared in Examples 3, 4, 5, and 6, respectively, and then further diluted to 0.2 mg / ml using PBS. Five aliquots of each of the five chloroacetamide + cysteine-monoclonal antibodies were dispensed and stored at 4°C for 12 h, 37°C for 24 h, 37°C for 72 h, and 37°C for 168 h for stability testing. A total of 25 storage conditions were applied for each of the five chloroacetamide + cysteine-monoclonal antibodies.

[0063] Monoclonal antibodies for 25 different conditions were diluted 100,000 times with PBS buffer. 100 μl of each antibody was added to an ELISA plate pre-coated with the corresponding detection protein. After incubation at 37°C for 1 hour, the plate was washed 3 times. Then, 100 μl of HRP-labeled goat anti-mouse secondary antibody (Jackson ImmunoResearch Inc., catalog number: 115-035-003) was added to each well. After incubation at 37°C for 1 hour, the plate was washed 3 times. Then, 100 μl of TMB substrate was added to each well, and the plate was incubated at 37°C for 5 minutes. Finally, 50 μl of stop solution was added to each well, and the OD value was measured at 450 nm using an ELISA reader.

[0064] The OD values ​​measured at 37℃ for 12h, 24h, 72h, and 168h, respectively, were compared with the OD value at 4℃ as the activity retention values ​​for each chloroacetamide + cysteine-monoclonal antibody.

[0065] The results are shown in Table 4. The chloroacetamide + cysteine-monoclonal antibody diluted 4 times with the protective solution showed the best stability. After 37℃ for 168h, the activity of the protected chloroacetamide + cysteine-monoclonal antibody was 98% retained; the activity of the chloroacetamide + cysteine-monoclonal antibody diluted with PBS was 89%.

[0066] Table 4.

[0067] For any points not covered above, existing technologies shall apply.

[0068] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for improving the stability of monoclonal antibodies containing free thiol groups, characterized in that, Chloroacetamide and cysteine ​​are used to prepare a reagent containing a free thiol monoclonal antibody, wherein the reagent contains chloroacetamide, cysteine ​​and a free thiol monoclonal antibody.

2. The method according to claim 1, characterized in that, In the reagent containing free thiol monoclonal antibody, the mass percentage of chloroacetamide is 0.005%~0.02%, and the mass percentage of cysteine ​​is 0.005%~0.02%.

3. The method according to claim 1, characterized in that, The reagent containing the free thiol monoclonal antibody also includes a protective solution prepared from a buffer, bovine serum albumin, cyclodextrin, arginine, and Proclin 300.

4. The method according to claim 3, characterized in that, The buffer solution is a phosphate buffer.

5. The method according to claim 4, characterized in that, The protective solution, by weight, comprises phosphate buffer, pH 7.4, 0.05 mol / L; bovine serum albumin 2-10 g / L; cyclodextrin 0.5-2 g / L; arginine 0.01 mol / L-0.02 mol / L; and Proclin 300 0.5-1 g / L.

6. The method according to claim 4, characterized in that, The protective solution, by weight, comprises phosphate buffer, pH 7.4, 0.05 mol / L; bovine serum albumin 2 g / L; cyclodextrin 2 g / L; arginine 0.01 mol / L; and Proclin 300 0.5 g / L.

7. The method according to claim 4, characterized in that, The protective solution, by weight, comprises phosphate buffer, pH 7.4, 0.05 mol / L; bovine serum albumin 2 g / L; cyclodextrin 1 g / L; arginine 0.02 mol / L; and Proclin 300 0.5 g / L.

8. The method according to claim 4, characterized in that, The protective solution, by weight, comprises phosphate buffer, pH 7.4, 0.05 mol / L; bovine serum albumin 5 g / L; cyclodextrin 1 g / L; arginine 0.01 mol / L; and Proclin 300 1 g / L.

9. The method according to claim 4, characterized in that, The protective solution, by weight, comprises PBS buffer, pH 7.4, 0.05 mol / L; bovine serum albumin 10 g / L; cyclodextrin 0.5 g / L; arginine 0.01 mol / L; and Proclin 300 g / L.

10. The use of the method according to any one of claims 1-9 in the preparation of a diagnostic kit.

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