Composite water-soluble immunologic adjuvant and preparation method thereof

Abstract

The invention discloses a composite water-soluble immunologic adjuvant and a preparation method thereof. The composite water-soluble immunologic adjuvant is prepared from the following components in parts by weight: 18-22 parts of phosphate buffer salt solution dry powder, 1-3 parts of sodium ascorbate, 8-12 parts of beta-glucan, 4-8 parts of xanthan gum and carrageenan mixture, 8-14 parts of plant extract, 2-5 parts of plant polysaccharide and 1930-1960 parts of ultrapure water. According to the adjuvant, the problem that a specific antibody is generated for the adjuvant is effectively avoided, the detection rate of positive clone is remarkably increased, and the possibility of obtaining a high-quality paired antibody is increased. The mixing process of the adjuvant and the water-soluble antigen is simple and does not need emulsification. The native conformation of the antigen is retained, and the generation of antibodies aiming at conformation-dependent epitopes is facilitated. Compared with traditional Freund's adjuvants and incomplete Freund's adjuvants, the composite water-soluble adjuvant has the advantages that the use amount of antigens is small, the immune cycle is shortened, efficient immune response and antibody generation can be quickly stimulated, and the composite water-soluble adjuvant has remarkable competitive advantages.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and vaccine technology, and specifically relates to a composite water-soluble adjuvant for rapid preparation of monoclonal antibodies or polyclonal antibodies and a preparation method thereof. Background Art

[0002] In the preparation of monoclonal or polyclonal antibodies, the selection of immune adjuvants is crucial because it directly affects the efficiency and quality of antibody production. In traditional antibody preparation technology, Freund's adjuvants (including complete Freund's adjuvant and incomplete Freund's adjuvant) are commonly used immune adjuvants, which can enhance the immunogenicity of antigens, but have some limitations, such as:

[0003] (1) Freund's complete adjuvant contains BCG, a strong immunogen that may cause animals to produce antibodies against BCG, thereby affecting the immune response to the target antigen.

[0004] (2) Freund's adjuvant is an oily adjuvant, which requires a tedious emulsification process when mixed with water-soluble antigens, which may destroy the conformation of the antigen.

[0005] (3) Traditional immunization cycles are long and require multiple immunizations, which causes great harm to animals and is not in line with animal welfare. Summary of the invention

[0006] The present invention aims at the deficiencies of the prior art, such as the specific antibodies that may be caused by traditional adjuvants, the complexity of the mixing process of antigens and adjuvants, and the destruction of the antigen conformation, and proposes a new solution, providing a composite water-soluble immune adjuvant and a preparation method thereof. The adjuvant of the present invention can stimulate immune response faster under the same conditions and is more outstanding in improving antibody titer. The adjuvant is used for rapid preparation of monoclonal antibodies or polyclonal antibodies with the characteristics of high efficiency, safety and simplicity.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] A composite water-soluble immune adjuvant is prepared from the following components by weight: 18-22 parts of phosphate buffered saline solution dry powder, 1-3 parts of sodium ascorbate, 8-12 parts of beta-glucan, 4-8 parts of a mixture of xanthan gum and carrageenan, 8-14 parts of a plant extract, 2-5 parts of a plant polysaccharide, and 1930-1960 parts of ultrapure water.

[0009] Preferably, the above-mentioned composite water-soluble immune adjuvant is made of the following components by weight: 18-22 parts of phosphate buffered saline powder, 1-2 parts of sodium ascorbate, 10-12 parts of β-glucan, 4-5 parts of a mixture of xanthan gum and carrageenan, 10-12 parts of plant extracts, 4-5 parts of plant polysaccharides, and 1930-1960 parts of ultrapure water.

[0010] Most preferably, the above-mentioned composite water-soluble immune adjuvant is made of the following components, by weight: 18-22 parts of phosphate buffered saline solution dry powder, 1-2 parts of sodium ascorbate, 10 parts of β-glucan, 5 parts of a mixture of xanthan gum and carrageenan, 12 parts of plant extracts, 4 parts of plant polysaccharides, and 1930-1960 parts of ultrapure water.

[0011] Preferably, the plant extract is one of pine bark extract, alfalfa extract and eucommia leaf extract. Most preferably, the plant extract is pine bark extract.

[0012] Preferably, the plant polysaccharide is one of Ganoderma lucidum polysaccharide, Astragalus polysaccharide and Radix Isatidis polysaccharide, preferably Ganoderma lucidum polysaccharide.

[0013] The phosphate buffered saline solution powder comprises the following components in parts by weight: 15-18 parts of sodium chloride, 0.3-0.5 parts of potassium chloride, 2.4-3 parts of disodium hydrogen phosphate, and 0.3-0.5 parts of potassium dihydrogen phosphate.

[0014] Preferably, the weight ratio of the xanthan gum to carrageenan mixture is 1:0.5-2, preferably 1:0.5-1, and most preferably 1:1.

[0015] The preparation method of the above-mentioned composite water-soluble immune adjuvant comprises the following steps:

[0016] The phosphate buffered saline solution powder is made into a phosphate buffered saline solution with a pH of 7.2-7.4, and sodium ascorbate, β-glucan, a mixture of xanthan gum and carrageenan, a plant extract and a plant polysaccharide are sequentially added to the phosphate buffered saline solution, stirred until all the added ingredients are completely dissolved, and filtered for sterilization. The obtained sterile composite solution is stored in an environment of 2 to 8° C. for subsequent use.

[0017] The phosphate buffered saline solution is specifically prepared by the following method: sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate are added to ultrapure water in proportion; then, the mixed solution is stirred with a magnetic stirrer until each component is completely dissolved, and the pH of the phosphate buffered saline solution is adjusted to 7.2-7.4 with hydrochloric acid and sodium hydroxide.

[0018] During the preparation process of the composite water-soluble immune adjuvant of the present invention, the uniformity and stability of the adjuvant are ensured by accurately controlling the ratio of each component and the stirring conditions, thereby improving the repeatability of the immune effect.

[0019] The composite water-soluble immunoadjuvant of the present invention comprises a plant extract and a plant polysaccharide as its core component. The plant extract comprises one of pine bark, alfalfa and eucommia leaf extract; and the plant polysaccharide comprises one of ganoderma lucidum, astragalus and isatis root polysaccharide. Among them, the active substances of pine bark extract mainly comprise chemical active substances such as volatile oils (such as α-pinene), polyphenols (such as gallic acid) and flavonoids (such as quercetin), which give it multiple biological activities such as antioxidant, antibacterial and anti-inflammatory. The chemical components of alfalfa extract mainly comprise triterpenoid saponins (such as alfalfa acid type triterpenoid saponins), flavonoids (such as apigenin), coumarins (such as simple coumarins) and alkaloids, etc., which have antioxidant and immunomodulatory effects. Eucommia leaf extract mainly contains chlorogenic acid, flavonoid compounds, phenolic (acid) compounds and terpenoid compounds, etc., and has pharmaceutical effects such as antibacterial, anti-inflammatory, antioxidant and improving animal immunity. Ganoderma lucidum polysaccharides, astragalus polysaccharides and isatis indigotica polysaccharides were selected because of their immunomodulatory properties, biocompatibility, biodegradability, low toxicity and safety.

[0020] The combination of the extract and polysaccharide in the adjuvant of the present invention can not only enhance the immunogenicity of the vaccine, improve the humoral and cellular immune responses, but also provide lasting immunity.

[0021] The composite water-soluble adjuvant of the present invention can overcome the limitations of traditional Freund's adjuvant and has the following characteristics:

[0022] (1) Safety: The composite water-soluble immune adjuvant of the present invention does not contain toxic components, such as inactivated Mycobacterium tuberculosis, thereby avoiding the side effects and specific antibody problems that may be caused by traditional adjuvants.

[0023] (2) High efficiency: Compared with traditional Freund's adjuvant, the adjuvant of the present invention can induce mice to produce higher titers of immune serum antibodies within the same period of time, showing stronger immunogenicity.

[0024] (3) Simplicity: The adjuvant is easy to mix with the water-soluble antigen without the need for complicated emulsification steps. Uniform mixing can be achieved by simple pipetting, which retains the natural conformation of the antigen and is conducive to the production of high-quality antibodies.

[0025] (4) Economical: The use of the adjuvant of the present invention can reduce the amount of antigen used, shorten the immunization cycle, reduce costs, and quickly stimulate an efficient immune response.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: the composite water-soluble immune adjuvant of the present invention has significant competitive advantages in the field of monoclonal antibody or polyclonal antibody preparation. Its components are non-toxic and do not contain inactivated Mycobacterium tuberculosis, which effectively avoids the problem of producing specific antibodies to the adjuvant. The mixing process of the adjuvant and the water-soluble antigen is simple, and uniform mixing can be achieved by simply blowing 5 to 10 times, without the need for complex emulsification steps. This method retains the natural conformation of the antigen, which is conducive to the production of antibodies against conformation-dependent epitopes. Compared with traditional Freund's adjuvant and its incomplete Freund's adjuvant, the composite water-soluble immune adjuvant of the present invention uses less antigen, shortens the immunization cycle, and can quickly stimulate efficient immune response and antibody production. It is expected to become an indispensable tool in scientific research and industrial production, which is of great significance for accelerating the research and development and production of antibodies, and has broad application prospects and commercial value. DETAILED DESCRIPTION

[0028] In the following embodiments, pine bark extract was purchased from Xi'an Ruierli Bioengineering Co., Ltd., China; alfalfa extract and Eucommia leaf extract were purchased from Shanyang Lianfeng Biotechnology Co., Ltd., China. KL-6 protein, PGI protein and PGII protein were purchased from Changzhou Aifukang Biotechnology Co., Ltd., China; CK-MB protein was purchased from Wuhan Yipu Biotechnology Co., Ltd., China; NGAL protein was purchased from Shanghai Xuanya Biotechnology Co., Ltd., China. Sodium ascorbate was purchased from Jiangsu Yuanzhiyuan Biotechnology Co., Ltd., China, CAS: 134-03-2; β-glucan was purchased from Shaanxi Rankang Biotechnology Co., Ltd., China, CAS: 160872-27-5; xanthan gum was purchased from Anhui Zhonghong Bioengineering Co., Ltd., China, CAS: 1168-42-9; carrageenan was purchased from Guangdong Mingcheng Biotechnology Co., Ltd., China, CAS: 9000-07-1. Ganoderma lucidum polysaccharide and astragalus polysaccharide were purchased from Xi'an An'ao Biotechnology Co., Ltd., China; and Radix Isatidis polysaccharide was purchased from Shaanxi Xiazhou Biotechnology Co., Ltd., China. Commercial water-soluble adjuvants were purchased from Suzhou Botlon Immunotechnology Co., Ltd., China, with the catalog number KX0210041. Freund's complete adjuvant was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., Germany, with the catalog number F5881; Freund's incomplete adjuvant was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., Germany, with the catalog number F5506.

[0029] The experimental methods described in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The parts described in the following examples are parts by weight, and the materials, reagents, etc. can be obtained from commercial sources unless otherwise specified.

[0030] Example 1

[0031] The preparation method of composite water-soluble adjuvant A, the specific preparation steps are as follows:

[0032] (1) Preparation of phosphate buffered saline: Accurately weigh 16 parts of sodium chloride, 0.4 parts of potassium chloride, 2.9 parts of disodium hydrogen phosphate and 0.5 parts of potassium dihydrogen phosphate, and add them to 1947.2 parts of ultrapure water in sequence; then, stir the mixed solution with a magnetic stirrer until all components are completely dissolved.

[0033] (2) pH adjustment: The pH of the phosphate buffer solution obtained in step (1) is adjusted using hydrochloric acid and sodium hydroxide to stabilize it in the range of 7.2 to 7.4.

[0034] (3) Preparation of composite solution: To the phosphate buffered saline solution obtained in step (2), add 2 parts of sodium ascorbate, 10 parts of β-glucan, 5 parts of a mixture of xanthan gum and carrageenan (weight ratio of 1:1), 12 parts of pine bark extract and 4 parts of Ganoderma lucidum polysaccharide in sequence; stir again using a magnetic stirrer until all the added ingredients are completely dissolved.

[0035] (4) Filtration and sterilization: The composite solution obtained in step (3) is filtered and sterilized to ensure the sterility of the solution.

[0036] (5) Storage: The sterile compound solution obtained in step (4) is stored in an environment of 2 to 8° C. for subsequent use.

[0037] The present invention can effectively prepare the composite water-soluble adjuvant A through the above method.

[0038] Example 2

[0039] The preparation method of the composite water-soluble adjuvant B, the specific preparation steps are as follows:

[0040] (1) Preparation of phosphate buffered saline: 17 parts of sodium chloride, 0.3 parts of potassium chloride, 2.5 parts of disodium hydrogen phosphate and 0.4 parts of potassium dihydrogen phosphate were accurately weighed and added to 1947.3 parts of ultrapure water in sequence; then, the mixed solution was stirred using a magnetic stirrer until all components were completely dissolved.

[0041] (2) pH adjustment: The pH of the phosphate buffer solution obtained in step (1) is adjusted using hydrochloric acid and sodium hydroxide to stabilize it in the range of 7.2 to 7.4.

[0042] (3) Preparation of composite solution: To the phosphate buffered saline solution obtained in step (2), 1.5 parts of sodium ascorbate, 12 parts of β-glucan, 4 parts of a mixture of xanthan gum and carrageenan (weight ratio of 1:0.5), 10 parts of pine bark extract and 5 parts of astragalus polysaccharide were added in sequence; the mixture was stirred again using a magnetic stirrer until all the added ingredients were completely dissolved.

[0043] (4) Filtration and sterilization: The composite solution obtained in step (3) is filtered and sterilized to ensure the sterility of the solution.

[0044] (5) Storage: The sterile compound solution obtained in step (4) is stored in an environment of 2 to 8° C. for subsequent use.

[0045] The present invention can effectively prepare the composite water-soluble adjuvant B through the above method.

[0046] Example 3

[0047] The preparation method of the composite water-soluble adjuvant C, the specific preparation steps are as follows:

[0048] (1) Preparation of phosphate buffered saline: Accurately weigh 18 parts of sodium chloride, 0.4 parts of potassium chloride, 2.6 parts of disodium hydrogen phosphate and 0.3 parts of potassium dihydrogen phosphate, and add them to 1948.2 parts of ultrapure water in sequence; then, stir the mixed solution with a magnetic stirrer until all components are completely dissolved.

[0049] (2) pH adjustment: The pH of the phosphate buffer solution obtained in step (1) is adjusted using hydrochloric acid and sodium hydroxide to stabilize it in the range of 7.2 to 7.4.

[0050] (3) Preparation of composite solution: To the phosphate buffered saline solution obtained in step (2), 2.5 parts of sodium ascorbate, 8 parts of β-glucan, 8 parts of a mixture of xanthan gum and carrageenan (weight ratio of 1:2), 8 parts of alfalfa extract and 4 parts of Radix Isatidis polysaccharide were added in sequence; and the mixture was stirred again using a magnetic stirrer until all the added ingredients were completely dissolved.

[0051] (4) Filtration and sterilization: The composite solution obtained in step (3) is filtered and sterilized to ensure the sterility of the solution.

[0052] (5) Storage: The sterile compound solution obtained in step (4) is stored in an environment of 2 to 8° C. for subsequent use.

[0053] The present invention can effectively prepare the composite water-soluble adjuvant C through the above method.

[0054] Example 4

[0055] The preparation method of the composite water-soluble adjuvant D, the specific preparation steps are as follows:

[0056] (1) Preparation of phosphate buffered saline: Accurately weigh 16 parts of sodium chloride, 0.5 parts of potassium chloride, 2.8 parts of disodium hydrogen phosphate and 0.4 parts of potassium dihydrogen phosphate, and add them to 1945.3 parts of ultrapure water in sequence; then, stir the mixed solution with a magnetic stirrer until all components are completely dissolved.

[0057] (2) pH adjustment: The pH of the phosphate buffer solution obtained in step (1) is adjusted using hydrochloric acid and sodium hydroxide to stabilize it in the range of 7.2 to 7.4.

[0058] (3) Preparation of composite solution: To the phosphate buffered saline solution obtained in step (2), add 3 parts of sodium ascorbate, 9 parts of β-glucan, 6 parts of a mixture of xanthan gum and carrageenan (weight ratio of 1:1.5), 14 parts of eucommia leaf extract and 3 parts of ganoderma lucidum polysaccharide in sequence; stir again using a magnetic stirrer until all added ingredients are completely dissolved.

[0059] (4) Filtration and sterilization: The composite solution obtained in step (3) is filtered and sterilized to ensure the sterility of the solution.

[0060] (5) Storage: The sterile compound solution obtained in step (4) is stored in an environment of 2 to 8° C. for subsequent use.

[0061] The present invention can effectively prepare the composite water-soluble adjuvant D through the above method.

[0062] Example 5

[0063] The composite water-soluble adjuvant A prepared in Example 1, the composite water-soluble adjuvant B prepared in Example 2, the composite water-soluble adjuvant C prepared in Example 3, and the composite water-soluble adjuvant D prepared in Example 4 were mixed with protein antigens, mice were immunized, and the effects of each adjuvant on the antibody titer of mouse serum were detected. The specific steps are as follows:

[0064] (1) Antigen preparation: Krebs von den Lungen-6 (KL-6) protein was selected as the antigen. KL-6 protein is a sialylated sugar chain antigen and is often used as one of the sensitive indicators of lung injury. 20 μg of antigen was diluted with normal saline to twice the final concentration (prepared at a dosage of 50 μL per injection).

[0065] (2) Mixing antigen and adjuvant: Under sterile conditions, take out the composite water-soluble immune adjuvants A, B, C and D prepared by the present invention and mix them with the antigen at a volume ratio of 1:1. Use a pipette to gently pipette 5 to 10 times to achieve rapid and uniform mixing.

[0066] (3) Animal immunization: On day 0, mice were given a primary immunization by subcutaneous injection at multiple points, and a booster immunization was given in the same manner on day 21.

[0067] (4) Blood collection and titer determination: A small amount of tail blood was collected on days 0, 21, and 35, and the serum was separated. The indirect ELISA method was used to determine the antibody titer of the mouse immune serum. In order to ensure the accuracy and repeatability of the experimental results, the OD of the negative control well was 450 The value should be controlled below 0.2; the titer of the sample well is determined by OD 450 A value of 1 or more was used as the criterion to avoid false positive results.

[0068] The results are shown in Table 1. On day 21, the antibody titer of the immune serum of the composite water-soluble adjuvants A and B had reached 12800. On day 35, the antibody titer of the immune serum of the composite water-soluble adjuvant A had reached 102400. This shows that the combination of the components of the composite water-soluble adjuvant A has the most significant effect on increasing the antibody titer of the KL-6 protein.

[0069] Table 1 Comparison of antibody titers in mouse immune sera with different formulas of composite water-soluble adjuvants

[0070]

[0071] Example 6

[0072] The preparation method of the composite water-soluble adjuvant E, the specific preparation steps are as follows:

[0073] (1) Preparation of phosphate buffered saline: Accurately weigh 16 parts of sodium chloride, 0.4 parts of potassium chloride, 2.9 parts of disodium hydrogen phosphate and 0.5 parts of potassium dihydrogen phosphate, and add them to 1947.2 parts of ultrapure water in sequence; then, stir the mixed solution with a magnetic stirrer until all components are completely dissolved.

[0074] (2) pH adjustment: The pH of the phosphate buffer solution obtained in step (1) is adjusted using hydrochloric acid and sodium hydroxide to stabilize it in the range of 7.2 to 7.4.

[0075] (3) Preparation of the composite solution: To the phosphate buffered saline solution obtained in step (2), 2 parts of sodium ascorbate, 10 parts of β-glucan, 5 parts of a mixture of xanthan gum and carrageenan (weight ratio of 1:1), 12 parts of pine bark extract and 4 parts of ultrapure water were added in sequence; the mixture was stirred again using a magnetic stirrer until all the added ingredients were completely dissolved.

[0076] (4) Filtration and sterilization: The composite solution obtained in step (3) is filtered and sterilized to ensure the sterility of the solution.

[0077] (5) Storage: The sterile compound solution obtained in step (4) is stored in an environment of 2 to 8° C. for subsequent use.

[0078] The present invention can effectively prepare the composite water-soluble adjuvant E through the above method.

[0079] Example 7

[0080] The preparation method of the composite water-soluble adjuvant F, the specific preparation steps are as follows:

[0081] (1) Preparation of phosphate buffered saline: Accurately weigh 16 parts of sodium chloride, 0.4 parts of potassium chloride, 2.9 parts of disodium hydrogen phosphate and 0.5 parts of potassium dihydrogen phosphate, and add them to 1947.2 parts of ultrapure water in sequence; then, stir the mixed solution with a magnetic stirrer until all components are completely dissolved.

[0082] (2) pH adjustment: The pH of the phosphate buffer solution obtained in step (1) is adjusted using hydrochloric acid and sodium hydroxide to stabilize it in the range of 7.2 to 7.4.

[0083] (3) Preparation of composite solution: To the phosphate buffered saline solution obtained in step (2), add 2 parts of sodium ascorbate, 10 parts of β-glucan, 5 parts of a mixture of xanthan gum and carrageenan (weight ratio of 1:1), 12 parts of ultrapure water and 4 parts of Ganoderma lucidum polysaccharide in sequence; stir again using a magnetic stirrer until all the added ingredients are completely dissolved.

[0084] (4) Filtration and sterilization: The composite solution obtained in step (3) is filtered and sterilized to ensure the sterility of the solution.

[0085] (5) Storage: The sterile compound solution obtained in step (4) is stored in an environment of 2 to 8° C. for subsequent use.

[0086] The present invention can effectively prepare the composite water-soluble adjuvant F through the above method.

[0087] Example 8

[0088] The composite water-soluble adjuvant A prepared in Example 1, the composite water-soluble adjuvant E prepared in Example 6, and the composite water-soluble adjuvant F prepared in Example 7 were mixed with protein antigens to immunize mice, and the effect of each adjuvant on the antibody titer of mouse serum was detected. The specific steps were the same as those in Example 5 (using KL-6 protein as the antigen).

[0089] The results are shown in Table 2. On the 21st day, the antibody titer of the mouse immune serum of the composite water-soluble adjuvants A and E had reached 12800, while the antibody titer of the mouse immune serum of the composite water-soluble adjuvant F only reached 3200. On the 35th day, the antibody titer of the mouse immune serum of the composite water-soluble adjuvant A had reached 102400, and the antibody titers of the mouse immune serum of the composite water-soluble adjuvants E and F were 51200 and 6400, respectively. This shows that the pine bark extract in the composite water-soluble adjuvant A is the most critical for improving the antibody titer of KL-6 protein. However, the combination with Ganoderma lucidum polysaccharide can produce a significant synergistic effect, thereby improving the antibody titer of mouse immune serum.

[0090] Table 2 Comparison of antibody titers in mouse immune sera of composite water-soluble adjuvants with different formulations

[0091]

[0092] Example 9

[0093] The composite water-soluble adjuvant A prepared in Example 1 of the present invention, commercial water-soluble adjuvants and traditional Freund's adjuvants (including Freund's complete adjuvant and Freund's incomplete adjuvant) were mixed with several protein antigens, mice were immunized, and the effects of each adjuvant on the antibody titer of mouse serum were comprehensively evaluated.

[0094] KL-6 protein, PGI protein, PGII protein, CK-MB protein and NGAL protein were used as antigens respectively.

[0095] Among them, pepsinogen I (PGI) and pepsinogen II (PGII) are precursor proteins secreted by gastric mucosal cells, involved in the digestion process, and have important clinical significance in the screening of gastric cancer; creatine kinase isoenzyme MB (CK-MB) is one of the specific serum markers of myocardial cell damage; neutrophil gelatinase-associated lipocalin (NGAL) is one of the most effective biological markers for diagnosing acute kidney injury and is also one of the effective markers for early diabetic nephropathy.

[0096] The immunization dosage and operation steps of the composite water-soluble adjuvant A prepared in Example 1 and the commercial water-soluble adjuvant are the same as those in Example 5.

[0097] The immunization dosage and operation steps of traditional Freund's adjuvant are as follows:

[0098] (1) Antigen preparation: Use sterile saline to dilute 100 μg of protein antigen to a volume of 100 μL per injection.

[0099] (2) Mixing the antigen and adjuvant: Under sterile conditions, take out Freund's complete adjuvant and mix it with the antigen liquid diluted in step (1) in a volume ratio of 1:1; prepare an oil-in-water viscous emulsion by emulsification.

[0100] (3) Animal immunization: On day 0, mice were given a primary immunization using Freund's complete adjuvant by multiple subcutaneous injections. On days 14, 28, and 42, mice were given three booster immunizations using Freund's incomplete adjuvant in the same manner.

[0101] (4) Blood collection and titer determination: On days 0, 21, 35, and 52, a small amount of tail blood was collected, the serum was separated, and the antibody titer of the mouse immune serum was determined using the indirect ELISA method; the OD450 value of the negative control well was ensured to be controlled below 0.2, and the titer of the sample well was determined based on the OD450 value reaching or exceeding 1 to avoid false positive results.

[0102] The adjuvant of the embodiment of the present invention is compared with the antibody titer of the immune serum of commercial water-soluble adjuvant and Freund's adjuvant. The results are shown in Tables 3, 4, 5, 6, and 7. On the 35th day, the composite water-soluble adjuvant A prepared in Example 1 can significantly improve the serum antibody titer of 5 antigens. Compared with the immunoenhancing performance of traditional Freund's adjuvant, the composite water-soluble adjuvant A not only has a significantly higher final antibody titer than Freund's adjuvant, but also reduces the amount of antigen used and shortens the immunization cycle. At the same time, there is no need for an emulsification step, which reduces protein loss and the destruction of the natural conformation of the antigen, thereby facilitating the production of antibodies against conformation-dependent epitopes. In addition, the composite water-soluble adjuvant A prepared in Example 1 is also better than commercial water-soluble adjuvants in improving the titer of antigen serum antibodies, showing its potential in commercial applications.

[0103] Table 3 Comparison of antibody titers of KL-6 protein mouse immune serum

[0104]

[0105] Table 4 Comparison of antibody titers of PGI protein mouse immune serum

[0106]

[0107]

[0108] Table 5 Comparison of antibody titers of PGII protein mouse immune serum

[0109]

[0110] Table 6 Comparison of CK-MB protein mouse immune serum antibody titer

[0111]

[0112] Table 7 Comparison of NGAL protein mouse immune serum antibody titer

[0113]

[0114] In summary, the composite water-soluble adjuvant provided by the present invention uses non-toxic ingredients and does not contain inactivated Mycobacterium tuberculosis, so that the adjuvant effectively avoids the problem of producing specific antibodies to the adjuvant, and also increases the possibility of obtaining high-quality paired antibodies. At the same time, the mixing process of the adjuvant and the water-soluble antigen is simple, and uniform mixing can be achieved by simply blowing and beating, without the need for complex emulsification steps, and the natural conformation of the antigen is effectively retained, which is conducive to the production of antibodies against conformation-dependent epitopes. In addition, compared to traditional Freund's adjuvant, the composite water-soluble adjuvant of the present invention uses less antigen amount, shortens the immune cycle, and can quickly stimulate efficient immune response and antibody production. Therefore, the composite water-soluble adjuvant provided by the present invention can quickly produce higher serum antibody titers, thereby laying a solid foundation for the preparation of monoclonal antibodies or polyclonal antibodies.

[0115] The composite water-soluble adjuvant of the present invention is applied in the field of monoclonal antibody or polyclonal antibody preparation, can significantly improve the efficiency and quality of antibody preparation, and becomes an important tool in scientific research and industrial production.

Claims

1. A composite water-soluble immune adjuvant, characterized in that The invention is prepared from the following components in parts by weight: 18-22 parts of phosphate buffered saline solution dry powder, 1-3 parts of sodium ascorbate, 8-12 parts of beta-glucan, 4-8 parts of a mixture of xanthan gum and carrageenan, 8-14 parts of a plant extract, 2-5 parts of a plant polysaccharide and 1930-1960 parts of ultrapure water.

2. The composite water-soluble immune adjuvant according to claim 1, characterized in that The invention is prepared from the following components in parts by weight: 18-22 parts of phosphate buffered saline solution dry powder, 1-2 parts of sodium ascorbate, 10-12 parts of beta-glucan, 4-5 parts of a mixture of xanthan gum and carrageenan, 10-12 parts of a plant extract, 4-5 parts of a plant polysaccharide and 1930-1960 parts of ultrapure water.

3. The composite water-soluble immune adjuvant according to claim 1 or 2, characterized in that: The phosphate buffered saline solution powder comprises the following components in parts by weight: 15-18 parts of sodium chloride, 0.3-0.5 parts of potassium chloride, 2.4-3 parts of disodium hydrogen phosphate, and 0.3-0.5 parts of potassium dihydrogen phosphate.

4. The composite water-soluble immune adjuvant according to claim 1 or 2, characterized in that: The weight ratio of the xanthan gum to carrageenan mixture is 1:0.5-2.

5. The composite water-soluble immune adjuvant according to claim 1 or 2, characterized in that: The plant extract is one of pine bark extract, alfalfa extract and eucommia leaf extract.

6. The composite water-soluble immune adjuvant according to claim 5, characterized in that: The plant extract is pine bark extract.

7. The composite water-soluble immune adjuvant according to claim 1 or 2, characterized in that: The plant polysaccharide is one of ganoderma lucidum polysaccharide, astragalus polysaccharide and isatis indigotica polysaccharide.

8. The composite water-soluble immune adjuvant according to claim 1 or 2, characterized in that: The plant polysaccharide is ganoderma lucidum polysaccharide.

9. A method for preparing the composite water-soluble immune adjuvant according to claim 1 or 2, characterized in that The method comprises the following steps: preparing a phosphate buffer saline solution with a pH of 7.2-7.4 from a phosphate buffer saline solution dry powder, sequentially adding sodium ascorbate, beta-glucan, a mixture of xanthan gum and carrageenan, a plant extract and a plant polysaccharide into the phosphate buffer saline solution, stirring until all the added components are completely dissolved, and filtering and sterilizing.

10. The method for preparing the composite water-soluble immune adjuvant according to claim 9, characterized in that The phosphate buffered saline solution is prepared by the following method: sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate are added into ultrapure water in sequence; then, the mixed solution is stirred by a magnetic stirrer until each component is completely dissolved, and the pH of the phosphate buffered saline solution is adjusted to 7.2-7.4 by using hydrochloric acid and sodium hydroxide.

Images