A kind of immune adjuvant and immunization method for preparing rabbit anti-CRP

Abstract

The present invention relates to the field of antibody technology, and proposes an immune adjuvant and an immunization method for preparing rabbit anti-CRP. The immune adjuvant comprises the following components: aluminum salt, kaempferol, dodecyl glucopyranoside, cyclodextrin package, ellagic acid, and deionized water. In the immune adjuvant of the present invention, the aluminum salt prolongs the retention time of the antigen at the injection site by adsorbing the antigen, thereby improving the uptake efficiency of antigen-presenting cells. The combination of kaempferol, cyclodextrin package, and ellagic acid can promote antigen presentation and the initiation of adaptive immunity by optimizing inflammation, enhancing antioxidant protection and immunomodulatory function, and accelerating the differentiation of plasma cells, thereby generating antibodies earlier. Dodecyl glucopyranoside effectively improves the uniformity of the system and the adsorption effect of each component, so that the synergistic mechanism of each component is fully utilized. The components in the immune adjuvant work together to achieve the technical effect of shortening the immunization cycle and improving the antibody titer.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to an immune adjuvant for preparing rabbit anti-CRP and an immunization method. Background Art

[0002] C-reactive protein (CRP) is a typical acute phase responder and an important regulator of immune responses. Under normal circumstances, CRP expression levels are very low, but after infection, its expression can increase by 10,000-fold within a few hours. Clinically, CRP is generally used as a preferred indicator for identifying bacterial or viral infections, and is used for the diagnosis and monitoring of autoimmune and infectious diseases, as well as for observing the efficacy of antibiotics.

[0003] The preparation of high-quality rabbit anti-CRP antibodies (such as polyclonal antibodies) is an important step in developing CRP-related detection reagents or in-depth research on its biological functions. In order to induce the body to produce specific antibodies against CRP, it is necessary to combine the CRP antigen with an immune adjuvant and improve the immunogenicity and titer of the antibody through a reasonable immunization method. Currently, Freund's adjuvant is commonly used as a classic immune adjuvant in the preparation of highly efficient and specific rabbit anti-CRP antibodies. The immunization plan usually requires a primary immunization and multiple booster immunizations, and the immunization cycle is generally 8-12 weeks, which is a long immunization cycle. In addition, the antigen and oily adjuvant need to be emulsified when mixed. During this operation, the natural conformation of the antigen is destroyed, making it difficult to obtain antibodies targeting structural epitopes. At the same time, the degree of emulsification is difficult to control consistently, causing large fluctuations in the immune effect, thereby affecting the immune effect and reducing the antibody titer. Summary of the Invention

[0004] In view of this, the present invention proposes a rabbit anti-CRP immune adjuvant and an immunization method with a short immunization cycle and no need for emulsification.

[0005] The technical solution of the present invention is achieved as follows: In the first aspect, the present invention provides an immune adjuvant for preparing rabbit anti-CRP, comprising the following components: aluminum salt, kaempferol, dodecyl glucopyranoside, cyclodextrin, ellagic acid and deionized water.

[0006] Aluminum salts prolong the retention time of antigens at the injection site by adsorbing antigens, thereby improving the uptake efficiency of antigen-presenting cells. Kaempferol can enhance the activity of antigen-presenting cells, while cyclodextrin and ellagic acid create a controlled microenvironment through their antioxidant and anti-inflammatory functions, protecting antigen-presenting cells from participating in immune regulation. The combination of the two can promote antigen presentation and the initiation of adaptive immunity by optimizing inflammation, enhancing antioxidant protection and immunomodulatory functions, and accelerating the differentiation of plasma cells, thereby generating antibodies earlier. Dodecylpyranoside effectively improves the uniformity of the system and the adsorption of each component, ensuring that aluminum salts, kaempferol, cyclodextrin and ellagic acid are reasonably dispersed within the system, allowing the synergistic mechanism to be fully utilized.

[0007] On the basis of the above technical solution, preferably, based on the mass concentration of 100%, the mass concentration of each component in the immune adjuvant is: aluminum salt 0.1%-0.5%, kaempferol 0.05%-0.3%, dodecyl glucopyranoside 0.01%-0.1%, cyclodextrin and ellagic acid 0.1%-0.3%, and the balance is deionized water.

[0008] Based on the above technical solution, preferably, the method of preparing the cyclodextrin package and ellagic acid is as follows: dissolving hydroxypropyl-β-cyclodextrin in a PBS buffer solution with a pH of 7.5-8.0, then adding ellagic acid, and continuing to stir at 40-50° C. until a clear solution is formed, followed by freeze-drying to obtain the cyclodextrin package and ellagic acid powder.

[0009] On the basis of the above technical solution, preferably, the mass ratio of hydroxypropyl-β-cyclodextrin:ellagic acid is 5-6:1.

[0010] On the basis of the above technical solution, preferably, the kaempferol is chemically modified with MPEG-NH2.

[0011] Original kaempferol is easily metabolized and degraded, resulting in a short duration of action, which indirectly reduces its immunomodulatory efficacy. Modifications have given kaempferol a "long-lasting" effect, extending its duration of action and thus providing sustained immune regulation throughout the immune response cycle. The introduction of hydrophilic polyethylene glycol (PEG) chains also enhances kaempferol's water solubility, allowing it to be evenly distributed within the immune adjuvant system.

[0012] In addition, the modified kaempferol also increases its synergistic ability with other components: after the solubility and dispersibility of kaempferol are improved, it can bind to aluminum salt particles more efficiently, further enhancing the antigen presentation function. The long-lasting antioxidant and anti-inflammatory effects of cyclodextrin and ellagic acid form a "double protection" with the long-lasting kaempferol, further promoting the activity of presenting cells and effector cells, as well as the body's immune response, enabling it to produce antibodies faster and obtain antibodies with higher affinity. In addition, the introduction of polyethylene glycol chains increases the hydrophilicity and stability of the system, reduces the risk of uneven distribution between components (such as precipitation and agglomeration), and further enhances the overall stability and immune-enhancing effect of the adjuvant.

[0013] On the basis of the above technical solution, preferably, the preparation method of the MPEG-NH2 modified kaempferol comprises the following steps:

[0014] S1, dissolving kaempferol in an organic solvent, then adding EDC and NHS, adjusting the pH to 7.5-8.5, and stirring for 20-30 minutes to obtain an activated kaempferol solution;

[0015] S2, dissolving MPEG-NH2 in an organic solvent, then slowly adding the activated kaempferol solution dropwise to the MPEG-NH2 solution at 30-40°C with stirring for 6-12 hours, maintaining the pH at 7.5-8.0. After the reaction is completed, the organic solvent is removed by rotary evaporation to obtain MPEG-NH2 modified kaempferol.

[0016] Based on the above technical solution, preferably, in step S1, the mass ratio of kaempferol:EDC:NHS is 1:1.2-1.5:1-1.2.

[0017] On the basis of the above technical solution, preferably, in step S2, the mass ratio of MPEG-NH2:kaempferol is 1.2-2:1.

[0018] Based on the above technical solution, preferably, the aluminum salt is one of aluminum hydroxide, aluminum phosphate and aluminum sulfate; and the organic solvent is DMF or DMSO.

[0019] In a second aspect, the present invention provides a method for preparing a rabbit anti-CRP immune adjuvant, comprising the following steps:

[0020] Dodecylpyranoglucoside was added to deionized water and stirred to dissolve, followed by adding aluminum salt and stirring until dissolved, and then adding MPEG-NH2 modified kaempferol under stirring, and after dissolution, cyclodextrin and ellagic acid were added, and stirred until dissolved to obtain a rabbit anti-CRP immune adjuvant.

[0021] In a third aspect, the present invention provides a method for immunizing an animal using the above-mentioned immune adjuvant, comprising the following steps:

[0022] S1, first immunization: the antigen is mixed with the immune adjuvant and injected subcutaneously at the back of the neck to immunize the animal;

[0023] S2, second immunization: 16-18 days after the first immunization, the same amount of antigen and immune adjuvant as the first immunization is used to immunize the animal subcutaneously at the back of the neck;

[0024] S3, blood was collected from animals on day 28 to test titer;

[0025] The antigen and the immune adjuvant are mixed in equal volume or mass.

[0026] The immune adjuvant and immunization method for preparing rabbit anti-CRP of the present invention have the following beneficial effects compared with the prior art:

[0027] In the immune adjuvant of the present invention, aluminum salt prolongs the retention time of antigen at the injection site by adsorbing antigen, thereby improving the uptake efficiency of antigen-presenting cells; dodecylpyranoglucoside improves the dispersibility of antigen and adjuvant, prevents the aggregation of aluminum salt particles, optimizes the stability of antigen presentation, and also enhances the binding of antigen and aluminum salt. The cyclodextrin package, ellagic acid, and kaempferol work together to rapidly activate and proliferate sensitized lymphocytes and accelerate their differentiation into plasma cells, thereby producing highly effective antibodies earlier and significantly shortening the immune cycle. The cyclodextrin package and ellagic acid quickly activate adaptive immunity, and kaempferol regulates and optimizes the microenvironment. Together, they can shorten the immune time to 28 days or less.

[0028] After modification with MPEG-NH2, kaempferol improves its solubility and dispersibility, allowing it to more efficiently bind to aluminum salt particles, further enhancing antigen presentation. The long-lasting antioxidant and anti-inflammatory effects of cyclodextrin and ellagic acid, combined with the long-acting kaempferol, provide a "double protection" that further promotes the activity of presenting and effector cells, as well as the body's immune response, enabling faster antibody production and higher-affinity antibodies. Furthermore, the introduction of polyethylene glycol chains increases the hydrophilicity and stability of the system, reducing the risk of uneven distribution among components (such as precipitation and aggregation), further enhancing the adjuvant's overall stability and immune-enhancing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1Graph showing the effects of immune adjuvants in Examples and Comparative Examples on cellular immunity in CRP-immunized mice. DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Kaempferol (kaempferol-3,7-dirhamnoside) CAS: 482-38-2 was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. MPEG-NH2 (methoxypolyethylene glycol-amino) CAS: 80506-64-5, molecular weight 550, was purchased from Shenzhen Meiluo Technology Co., Ltd. Ellagic acid, CAS: 476-66-4, was purchased from Shaanxi Xinyanghe Biotechnology Co., Ltd.

[0033] Example 1

[0034] Taking the preparation of 1 L of immune adjuvant as an example, the immune adjuvant for preparing rabbit anti-CRP in this embodiment includes: 3.5 g / L of aluminum hydroxide, 1.5 g / L of kaempferol, 0.3 g / L of dodecyl glucopyranoside, 2 g / L of cyclodextrin and ellagic acid, and the balance is deionized water.

[0035] The preparation method of the immune adjuvant of this embodiment comprises the following steps:

[0036] S1, prepare kaempferol solution

[0037] Kaempferol is difficult to dissolve, so a solution must be prepared before mixing with other reagents. To prepare the kaempferol solution, weigh 1.5 g of kaempferol and add it to 50 mL of deionized water. Heat to 60°C and stir until completely dissolved. Cool to obtain the kaempferol solution.

[0038] S2, Cyclodextrin Packaging and Ellagic Acid Preparation

[0039] 15 g of hydroxypropyl-β-cyclodextrin was dissolved in 60 mL of 0.1 M pH 8.0 PBS buffer, and then 3 g of ellagic acid was added. Stirring was continued at 45 °C until a clear solution was formed, and then freeze-dried (pre-freezing at 50 °C for 6 h; primary drying at 20 Pa, -15 °C for 16 h; secondary drying at 25 °C for 10 h) to obtain cyclodextrin and ellagic acid powder.

[0040] S3, add 700 mL of 0.3 g of dodecyl pyranoside to deionized water and stir to dissolve it, then add 3.5 g of aluminum hydroxide and stir until dissolved, then add the kaempferol solution of step S1 under stirring, add 2 g of cyclodextrin and ellagic acid after dissolution, add deionized water to make the volume 1 L, and stir until dissolved to obtain a rabbit anti-CRP immune adjuvant.

[0041] Example 2

[0042] Taking the preparation of 1L of immune adjuvant as an example, the immune adjuvant for preparing rabbit anti-CRP in this embodiment includes: 3.5g / L of aluminum hydroxide, 1.5g / L of MPEG-NH2 modified kaempferol, 0.3g / L of dodecyl glucopyranoside, 2g / L of cyclodextrin and ellagic acid, and the balance is deionized water.

[0043] The preparation method of the immune adjuvant of this embodiment comprises the following steps:

[0044] S1, Preparation of MPEG-NH2 modified kaempferol

[0045] S11, dissolving 3 g of kaempferol in 60 mL of an organic solvent, DMF, followed by adding 4 g of EDC and 3 g of NHS, adjusting the pH to 7.5, and stirring the mixture for 25 min to obtain an activated kaempferol solution;

[0046] S12, dissolving 4.5 g of MPEG-NH2 in 80 mL of the organic solvent DMF, and then slowly adding the activated kaempferol solution in step S11 dropwise to the MPEG-NH2 solution, stirring at 35° C. for 10 h, maintaining the pH at 7.5. After the reaction is completed, the organic solvent is removed by rotary evaporation to obtain MPEG-NH2-modified kaempferol.

[0047] S2, Cyclodextrin Packaging and Ellagic Acid Preparation

[0048] 15 g of hydroxypropyl-β-cyclodextrin was dissolved in 100 mL of 0.1 M pH 8.0 PBS buffer, and then 3 g of ellagic acid was added. Stirring was continued at 45°C until a clear solution was formed, and then freeze-dried (pre-freezing at 50°C for 6 h; primary drying at 20 Pa, -15°C for 16 h; secondary drying at 25°C for 10 h) to obtain cyclodextrin and ellagic acid powder.

[0049] S3. Add 600 mL of 0.3 g of dodecylpyranoside to deionized water and stir to dissolve it. Then, add 3.5 g of aluminum hydroxide and stir until dissolved. Then, add 1.5 g of MPEG-NH2-modified kaempferol while stirring. After dissolution, add 2 g of cyclodextrin and ellagic acid. Add deionized water to make the volume to 1 L and stir until dissolved to obtain a rabbit anti-CRP immune adjuvant.

[0050] Example 3

[0051] Taking the preparation of 1L of immune adjuvant as an example, the immune adjuvant for preparing rabbit anti-CRP in this embodiment includes: 1g / L aluminum phosphate, 2g / L MPEG-NH2 modified kaempferol, 0.8g / L dodecyl glucopyranoside, 3g / L cyclodextrin and ellagic acid, and the balance is deionized water.

[0052] The preparation method of the immune adjuvant of this embodiment comprises the following steps:

[0053] S1, Preparation of MPEG-NH2 modified kaempferol

[0054] S11, dissolving 3 g of kaempferol in 60 mL of the organic solvent DMSO, then adding 3.6 g of EDC and 3.6 g of NHS, adjusting the pH to 8.5, and stirring the mixture for 20 min to obtain an activated kaempferol solution;

[0055] S12, dissolving 3.6 g of MPEG-NH2 in 80 mL of the organic solvent DMSO, and then slowly adding the activated kaempferol solution in step S11 dropwise to the MPEG-NH2 solution at 30°C with stirring for 12 h, with the pH maintained at 8.0. After the reaction is completed, the organic solvent is removed by rotary evaporation to obtain MPEG-NH2-modified kaempferol.

[0056] S2, Cyclodextrin Packaging and Ellagic Acid Preparation

[0057] 18 g of hydroxypropyl-β-cyclodextrin was dissolved in 180 mL of PBS buffer (pH 8.0), and then 3 g of ellagic acid was added. Stirring was continued at 50°C until a clear solution was formed, and then freeze-dried (pre-freezing at 50°C for 6 h; primary drying at 20 Pa, -15°C for 16 h; secondary drying at 25°C for 10 h) to obtain cyclodextrin and ellagic acid powder.

[0058] S3. Add 0.8 g of dodecylpyranoside to 700 mL of deionized water and stir to dissolve it. Then add 1 g of aluminum phosphate and stir until dissolved. Then, add 2 g of MPEG-NH2-modified kaempferol while stirring. After dissolution, add 3 g of cyclodextrin and ellagic acid. Add deionized water to make the volume to 1 L and stir until dissolved to obtain a rabbit anti-CRP immune adjuvant.

[0059] Example 4

[0060] Taking the preparation of 1L of immune adjuvant as an example, the immune adjuvant for preparing rabbit anti-CRP in this embodiment includes: 4g / L aluminum phosphate, 0.5g / L MPEG-NH2 modified kaempferol, 1g / L dodecyl glucopyranoside, 2.5g / L cyclodextrin and ellagic acid, and the balance is deionized water.

[0061] The preparation method of the immune adjuvant of this embodiment comprises the following steps:

[0062] S1, Preparation of MPEG-NH2 modified kaempferol

[0063] S11, dissolving 3 g of kaempferol in 60 mL of an organic solvent, DMF, followed by adding 3.5 g of EDC and 3.3 g of NHS, adjusting the pH to 8.5, and stirring the mixture for 30 min to obtain an activated kaempferol solution;

[0064] S12, dissolving 6 g of MPEG-NH2 in 80 mL of the organic solvent DMF, and then slowly adding the activated kaempferol solution in step S11 dropwise to the MPEG-NH2 solution at 40°C with stirring for 6 h, with the pH maintained at 8.0. After the reaction is completed, the organic solvent is removed by rotary evaporation to obtain MPEG-NH2-modified kaempferol.

[0065] S2, Cyclodextrin Packaging and Ellagic Acid Preparation

[0066] 16 g of hydroxypropyl-β-cyclodextrin was dissolved in 160 mL of 0.1 M pH 8.0 PBS buffer, and then 3 g of ellagic acid was added. Stirring was continued at 40°C until a clear solution was formed, and then freeze-dried (pre-freezing at 50°C for 6 h; primary drying at 20 Pa, -15°C for 16 h; secondary drying at 25°C for 10 h) to obtain cyclodextrin and ellagic acid powder.

[0067] S3. Add 1 g of dodecylpyranoside to 700 mL of deionized water and stir to dissolve it. Then, add 4 g of aluminum phosphate and stir until dissolved. Then, slowly add 0.5 g of MPEG-NH2-modified kaempferol dropwise while stirring. After dissolution, add 2.5 g of cyclodextrin and ellagic acid. Add deionized water to make the volume to 1 L and stir until dissolved to obtain a rabbit anti-CRP immune adjuvant.

[0068] Example 5

[0069] Taking the preparation of 1L of immune adjuvant as an example, the immune adjuvant for preparing rabbit anti-CRP in this embodiment includes: 5g / L of aluminum hydroxide, 3g / L of MPEG-NH2 modified kaempferol, 0.1g / L of dodecyl glucopyranoside, 1g / L of cyclodextrin and ellagic acid, and the balance is deionized water.

[0070] The preparation method of the immune adjuvant of this embodiment comprises the following steps:

[0071] S1, Preparation of MPEG-NH2 modified kaempferol

[0072] S11, dissolving 3 g of kaempferol in 60 mL of an organic solvent, DMF, followed by adding 4.5 g of EDC and 3.5 g of NHS, adjusting the pH to 8, and stirring the mixture for 20 min to obtain an activated kaempferol solution;

[0073] S12, dissolving 5 g of MPEG-NH2 in 60-100 mL of an organic solvent, DMF or DMSO, and then slowly adding the activated kaempferol solution obtained in step S11 dropwise to the MPEG-NH2 solution at 35° C. with stirring for 8 h, maintaining the pH at 7.5. After the reaction is completed, the organic solvent is removed by rotary evaporation to obtain MPEG-NH2-modified kaempferol.

[0074] S2, Cyclodextrin Packaging and Ellagic Acid Preparation

[0075] 17 g of hydroxypropyl-β-cyclodextrin was dissolved in 170 mL of 0.1 M pH 7.5 PBS buffer, and then 3 g of ellagic acid was added. Stirring was continued at 50°C until a clear solution was formed, and then freeze-dried (pre-freezing at 50°C for 6 h; primary drying at 20 Pa, -15°C for 16 h; secondary drying at 25°C for 10 h) to obtain cyclodextrin and ellagic acid powder.

[0076] S3. Add 0.1 g of dodecylpyranoglucoside to deionized water and stir to dissolve it. Then add 5 g of aluminum hydroxide and stir until dissolved. Then, add 3 g of MPEG-NH2-modified kaempferol while stirring. After dissolution, add 1 g of cyclodextrin and ellagic acid. Add deionized water to make the volume to 1 L and stir until dissolved to obtain a rabbit anti-CRP immune adjuvant.

[0077] Comparative Example 1

[0078] Compared with Example 1, Comparative Example 1 lacks kaempferol as the immune adjuvant, and the rest of the contents are the same as Example 1.

[0079] Comparative Example 2

[0080] Compared with Example 1, the immune adjuvant in Comparative Example 2 lacks the cyclodextrin package and ellagic acid, and the rest of the contents are the same as Example 1.

[0081] Comparative Example 3

[0082] Compared with Example 1, Comparative Example 3 lacks dodecyl glucopyranoside as the immune adjuvant, and the rest of the contents are the same as Example 1.

[0083] Comparative Example 4

[0084] Compared with Example 1, in Comparative Example 4, the concentration of kaempferol in the immune adjuvant exceeds the specified range, specifically 5 g / L. The rest of the contents are the same as in Example 1.

[0085] Comparative Example 5

[0086] Compared with Example 1, in Comparative Example 5, the concentrations of cyclodextrin and ellagic acid in the immune adjuvant exceeded the specified range, specifically 5 g / L. The rest of the contents were the same as in Example 1.

[0087] Comparative Example 6

[0088] Comparative Example 6 Compared with Example 2, in the MPEG-NH2-modified kaempferol, the concentrated amount of MPEG-NH2 is 2.5 times the weight of kaempferol, and the rest of the contents are the same as Example 2.

[0089] Immune titer testing

[0090] The immune adjuvants prepared in the above examples and comparative examples were used to immunize rabbits, with 3 mice used for each example or comparative example to verify the immune effect.

[0091] (1) The antigens and animal information selected for the immunization experiment are as follows:

[0092] The antigen was C Reactive Protein (Human), 2.47 mg / mL (OD at 280 nm), purchased from ADVY CHEMICAL PVT.LTD. Antigen storage medium: 50 mM Tris at pH 8.0 containing 200 mM NaCl, 5 mM CaCl, and 15 mM NaN₃.

[0093] Dilute the CRP antigen to a concentration of 1.2 mg / mL with 0.01 M PBS for later use.

[0094] Animals: Japanese large-eared white rabbits, 8-12 weeks old, female, purchased from Wuhan Wanqian Jiaxing Biotechnology Co., Ltd.

[0095] (2) Immunization plan:

[0096] 1. First immunization: Mix 100 μg antigen with 100 μg immune adjuvant (mass ratio 1:1), fix the rabbit, and inject subcutaneously at the back of the neck to immunize the animal;

[0097] 2. Second immunization: 17 days after the first immunization, the same amount of antigen and immune adjuvant as the first immunization is used to subcutaneously inject the immunized animal at the back of the neck;

[0098] 3. On day 28, blood was collected from the animals and the titer was tested using Elisa.

[0099] (3) Establishment of ELISA method for determining serum titer:

[0100] 1. Antigen coating: Coat the ELISA plate with CRP antigen (2 μg / mL) at 4°C overnight.

[0101] 2. Blocking: Add blocking solution (2% BSA), incubate at 37°C for 2 hours, then discard the blocking solution, pat dry, and dry at 37°C for 2 hours.

[0102] 3. Add the test serum: Dilute the test serum starting at 1:1000, and then dilute it 1:2 per well. Incubate at 37°C for 30 minutes, wash the plate (PBST), and pat dry.

[0103] 4. Add goat anti-rabbit enzyme-labeled antibody: dilute the goat anti-rabbit enzyme-labeled antibody to the working concentration (1:5000), incubate at 37°C for 30 minutes, wash the plate and pat dry.

[0104] 5. Color development: Add color developing solution (TMB), react at 37°C for 10 min, and measure the OD value using a dual-wavelength microplate reader.

[0105] Table 1 Absorbance values of Examples and Comparative Examples

[0106]

[0107] As shown in Table 1, the immunoadjuvants of the Examples of the present invention were all higher than those of the Comparative Example, specifically higher by 50W, demonstrating that the immunoadjuvants of the present invention are effective in increasing immune titer. As shown in Examples 1 and 2, the immunogenicity of kaempferol modified with MPEG-NH2 was significantly improved. This is because the modification of kaempferol with MPEG-NH2 not only increases its solubility and immunostimulatory efficacy, but also enhances the synergistic effect between the components.

[0108] Comparative Examples 1-3 show that the lack of any one of kaempferol, dodecyl glucopyranoside, cyclodextrin and ellagic acid will reduce the immune titer, which proves that the synergistic effect of each component has the effect of enhancing immunity.

[0109] From Comparative Examples 4-5, it can be seen that when the dosage of kaempferol, cyclodextrin and ellagic acid exceeds the specified range, the immune titer does not increase but decreases. This is because certain immune pathways may produce negative feedback regulation due to excessive activation, thereby inhibiting the immune titer; in addition, after exceeding the range, the synergistic effect is destroyed, resulting in a decrease in the immune effect.

[0110] Comparative Example 6 shows that when the amount of MPEG-NH2 exceeds the specified range, the immune titer is reduced. This may be because the excessive amount of MPEG-NH2 causes the molecular chain to be too long or too dense, forming steric hindrance and hindering the effective binding of kaempferol to the surface receptors of immune cells.

[0111] The second immunization of Example 1 was set on the 16th and 18th days, and the serum titer was measured according to the above method. The results are shown in Table 2.

[0112] Table 2 Effect of the second immunization time on serum titer

[0113]

[0114] As shown in Table 2, there was no significant difference in serum titer when immunized on days 16-18, and both could reach about 50W. In actual operation, the second immunization could be performed on any day between days 16-18.

[0115] 2. Irritation Index Test

[0116] To analyze the immune effect of the immune adjuvant, the present invention conducted the following experiment: after the immunization, the experimental rabbits were killed, the spleen was taken out to prepare the spleen cell suspension, and the proportion of living cells was ensured to be ≥95%. The spleen cell suspension was diluted to 1×10 7 The diluted spleen cell suspension was added to a 96-well plate (100 μL per well) and divided into three groups: 100 μL of concanavalin A (5 μg / mL), 100 μL of pokeweed (5 μg / mL), and 100 μL of phytohemagglutinin (5 μg / mL), with 5 wells per group. A control group was supplemented with 100 μL of 1640 medium. The cells were incubated at 37°C, 5% CO₂ for 68 h. Cell proliferation was then assessed using the MTT assay, and the stimulation index (SI) was calculated as the OD value of the mitogen-treated culture divided by the OD value of the mitogen-free culture. The results are shown in Table 3.

[0117] Table 3 Irritation index of Examples and Comparative Examples

[0118]

[0119] As shown in Table 3, compared with the comparative example, the immune adjuvant of the embodiment of the present invention can greatly enhance the proliferation response of spleen cells of CRP-immunized rabbits induced by three mitogens: ConA, PWM and PHA.

[0120] Comparative Examples 1-3 show that the synergistic effect of kaempferol, dodecyl glucopyranoside and cyclodextrin combined with ellagic acid enhances the cellular immune response of experimental rabbits to the immunogen. The absence of any of these components will weaken the cellular immune response, which is similar to the results in Table 1.

[0121] Comparative Examples 4-6 show that when the dosage of each component exceeds the specified range, negative feedback regulation will occur, inhibiting the immune response and thus causing a decrease in the intensity of the immune response.

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An immune adjuvant for preparing rabbit anti-CRP, characterized in that: The immunoadjuvant is composed of the following components based on a mass concentration of 100%: aluminum salt 0.1%-0.5%, kaempferol 0.05%-0.3%, dodecyl glucopyranoside 0.01%-0.1%, cyclodextrin and ellagic acid 0.1%-0.3%, and the balance is deionized water; The preparation method of the cyclodextrin bag and ellagic acid is as follows: dissolving hydroxypropyl-β-cyclodextrin in a PBS buffer solution with a pH of 7.5-8.0, then adding ellagic acid, stirring at 40-50° C. until a clear solution is formed, and then freeze-drying to obtain the cyclodextrin bag and ellagic acid powder; the mass ratio of the hydroxypropyl-β-cyclodextrin to ellagic acid is 5-6:1; The aluminum salt is one of aluminum hydroxide and aluminum phosphate.

2. The immune adjuvant for preparing rabbit anti-CRP according to claim 1, wherein: The kaempferol is chemically modified by MPEG-NH2, and the modification method comprises the following steps: S1, dissolving kaempferol in an organic solvent, then adding EDC and NHS, adjusting the pH to 7.5-8.5, and stirring for 20-30 minutes to obtain an activated kaempferol solution; S2, dissolving MPEG-NH2 in an organic solvent, then slowly adding the activated kaempferol solution dropwise to the MPEG-NH2 solution at 30-40°C while stirring for 6-12 hours, maintaining the pH at 7.5-8.

0. After the reaction is completed, the organic solvent is removed by rotary evaporation to obtain MPEG-NH2 modified kaempferol.

3. The immune adjuvant for preparing rabbit anti-CRP according to claim 2, characterized in that: In step S1, the mass ratio of kaempferol:EDC:NHS is 1:1.2-1.5:1-1.

2.

4. The immune adjuvant for preparing rabbit anti-CRP according to claim 2, characterized in that: In step S2, the mass ratio of MPEG-NH2:kaempferol is 1.2-2:

1.

5. The immune adjuvant for preparing rabbit anti-CRP according to claim 2, characterized in that: The organic solvent was DMF or DMSO.

6. The method for preparing a rabbit anti-CRP immune adjuvant according to any one of claims 2 to 5, characterized in that: The following steps are involved: Dodecylpyranoglucoside was added to deionized water and stirred to dissolve, followed by adding aluminum salt and stirring until dissolved, and then adding MPEG-NH2 modified kaempferol under stirring, and after dissolution, cyclodextrin and ellagic acid were added, and stirred until dissolved to obtain a rabbit anti-CRP immune adjuvant.

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