Immune adjuvant for preparing rabbit anti-CRP (C-reactive protein) and immunization method

Abstract

The invention relates to the technical field of antibodies, and provides an immunologic adjuvant for preparing rabbit anti-CRP (C-reactive protein) and an immunization method. The immunologic adjuvant is prepared from the following components: aluminum salt, kaempferitrin, dodecyl glucopyranoside, cyclodextrin inclusion, ellagic acid and deionized water. In the immunologic adjuvant, the aluminum salt adsorbs the antigen to prolong the residence time of the antigen at an injection site and improve the uptake efficiency of antigen presenting cells. The combination of the kaempferitrin, the cyclodextrin inclusion and the ellagic acid can promote antigen presentation and starting of adaptive immunity and accelerate differentiation of plasma cells by optimizing inflammation and enhancing antioxidant protection and immune regulation functions, so that an antibody is generated earlier. And 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 exerted. All the components in the immunologic adjuvant act together, so that the technical effects of shortening the immune cycle and improving the titer of the antibody are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and particularly 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 response protein and an important regulator of the immune resistance response. Under normal circumstances, the expression level of C-reactive protein is very low, but after being stimulated by infection, its expression level can increase by 10,000 times after a few hours. Clinically, CRP is generally used as a first-choice indicator for differentiating bacterial or viral infections, for the diagnosis and monitoring of autoimmune and infectious diseases, and for observing the efficacy of antibiotics, etc.

[0003] Preparing high-quality rabbit anti-CRP antibodies (such as polyclonal antibodies) is an important link in developing CRP-related detection reagents or deeply studying 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. At present, Freund's adjuvant is usually used as a classic immune adjuvant in the process of preparing highly specific rabbit anti-CRP antibodies. The immunization protocol usually requires primary immunization and multiple booster immunizations, and the immunization cycle is generally 8 - 12 weeks, with a relatively long immunization cycle; moreover, when the antigen and the oily adjuvant are mixed, emulsification is required, and the natural conformation of the antigen will be destroyed during the operation process, making it difficult to obtain antibodies against structural epitopes. At the same time, it is also difficult to control the emulsification degree consistently, resulting in 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 provides an immune adjuvant for rabbit anti-CRP with a short immunization cycle and no need for emulsification, as well as an immunization method.

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

[0006] Aluminum salts prolong the residence 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, and cyclodextrin-encapsulated ellagic acid creates a controlled microenvironment through antioxidant and anti-inflammatory functions to protect antigen-presenting cells from participating in immune regulation. The combination of the two can promote antigen presentation and the initiation of adaptive immunity, accelerate the differentiation of plasma cells, and thus generate antibodies earlier by optimizing inflammation, enhancing antioxidant protection, and immune regulatory functions. Dodecyl glucopyranoside effectively improves the uniformity of the system and the adsorption of each component, ensuring the reasonable dispersion of aluminum salts, kaempferol, and cyclodextrin-encapsulated ellagic acid in the system, and giving full play to the synergistic mechanism.

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

[0008] On the basis of the above technical solutions, preferably, the method for cyclodextrin-encapsulating ellagic acid is as follows: dissolve hydroxypropyl-β-cyclodextrin in PBS buffer solution with a pH of 7.5 - 8.0, then add ellagic acid, and continue stirring at 40 - 50 °C until a clear solution is formed, and then freeze-dry to obtain cyclodextrin-encapsulated ellagic acid powder.

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

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

[0011] The original kaempferol is easily metabolized and degraded, with a short duration of action, indirectly reducing its immune regulatory efficacy. After modification, the "long-acting" characteristic is imparted to kaempferol, prolonging the action time, and thus continuously supporting immune regulation throughout the immune response cycle. After introducing the hydrophilic polyethylene glycol (PEG) chain, the water solubility of kaempferol is also improved, enabling it to be evenly distributed in the immune adjuvant system.

[0012] In addition, the modified kaempferitrin also increases its synergistic ability with other components: after the solubility and dispersibility of kaempferitrin are improved, it can bind aluminum salt particles more efficiently, further enhancing the antigen presentation function. The long-term antioxidant and anti-inflammatory effects of cyclodextrin-encapsulated ellagic acid and the long-acting kaempferitrin form a "double protection", further promoting the activities of antigen-presenting cells and effector cells, as well as the immune response effect of the body, 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 (such as precipitation and aggregation) among components, and further enhances the overall stability and immune enhancement effect of the adjuvant.

[0013] On the basis of the above technical solutions, preferably, the preparation method of the MPEG-NH2-modified kaempferitrin includes the following steps: S1, Dissolve kaempferitrin in an organic solvent, then add EDC and NHS, and adjust the pH to 7.5 - 8.5, stir and react for 20 - 30 min to obtain an activated kaempferitrin solution; S2, Dissolve MPEG-NH2 in an organic solvent, and then slowly drop the activated kaempferitrin solution into the MPEG-NH2 solution, at 30 - 40 °C, stir while adding, stir for 6 - 12 h, maintain the pH at 7.5 - 8.0, and after the reaction, remove the organic solvent by rotary evaporation to obtain MPEG-NH2-modified kaempferitrin.

[0014] On the basis of the above technical solutions, preferably, in step S1, the mass ratio of kaempferitrin: EDC: NHS is 1: 1.2 - 1.5: 1 - 1.2.

[0015] On the basis of the above technical solutions, preferably, in step S2, the mass ratio of MPEG-NH2: kaempferitrin is 1.2 - 2: 1.

[0016] On the basis of the above technical solutions, preferably, the aluminum salt is one of aluminum hydroxide, aluminum phosphate, and aluminum sulfate; the organic solvent is DMF or DMSO.

[0017] In the second aspect, the present invention provides a preparation method of an immunoadjuvant for preparing rabbit anti-CRP, including the following steps: Add dodecyl glucopyranoside to deionized water and stir to dissolve it, then add an aluminum salt and stir until dissolved, and then add MPEG-NH2-modified kaempferitrin under stirring, and after dissolution, add cyclodextrin-encapsulated ellagic acid and stir until dissolved to obtain an immunoadjuvant for rabbit anti-CRP.

[0018] In the third aspect, the present invention provides a method for immunizing animals using the above immunoadjuvant, including the following steps: S1, First immunization: Mix the antigen with the immunoadjuvant and inject it subcutaneously into the back of the neck of the immunized animal; S2, Second immunization: On the 16th - 18th day after the first immunization, use the same dosage of antigen and immunoadjuvant as in the first immunization, and inject it subcutaneously into the back of the neck of the immunized animal; S3, Collect blood from the animal on the 28th day to detect the titer; The antigen and the immunoadjuvant are mixed in equal volume or equal mass.

[0019] A kind of immunoadjuvant for preparing rabbit anti - CRP and an immunization method of the present invention have the following beneficial effects compared with the prior art: In the immunoadjuvant of the present invention, aluminum salt prolongs the residence time of the antigen at the injection site by adsorbing the antigen, and improves the uptake efficiency of antigen - presenting cells; dodecylpyranoside improves the dispersibility of the antigen and the adjuvant, prevents the aggregation of aluminum salt particles, optimizes the stability of antigen presentation, and also enhances the binding of the antigen to the aluminum salt. Cyclodextrin - encapsulated ellagic acid and kaempferol act together to rapidly activate, proliferate sensitized lymphocytes, and accelerate their differentiation into plasma cells, thereby producing high - efficiency antibodies earlier, and significantly shortening the immune cycle. Cyclodextrin - encapsulated ellagic acid rapidly activates the adaptive immunity, and kaempferol regulates and optimizes the microenvironment. The two can shorten the immune time to 28 days or less.

[0020] After kaempferol is modified by MPEG - NH2, its solubility and dispersibility are improved, and it can bind aluminum salt particles more efficiently, further strengthening the antigen - presenting function. The long - acting antioxidant and anti - inflammatory effects of cyclodextrin - encapsulated ellagic acid and the long - acting kaempferol form a "double protection", further promoting the activities of antigen - presenting cells and effector cells, as well as the immune response effect of the body, enabling it to produce antibody ability 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 (such as precipitation and aggregation) between components, and further enhances the overall stability and immune enhancement effect of the adjuvant. Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 It is a diagram showing the influence of the immunoadjuvant of the embodiment and the comparative example on the cellular immunity of CRP - immunized mice. Detailed Embodiments

[0023] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0024] Kaempferitrin (kaempferol-3,7-dirhamnoside) CAS: 482-38-2 was purchased from Shanghai Macklin Biochemical Co., Ltd. MPEG-NH2 (methoxypolyethylene glycol-amine) CAS: 80506-64-5, with a molecular weight of 550, was purchased from Shenzhen Meiluo Technology Co., Ltd. Ellagic acid, CAS: 476-66-4, was purchased from Shaanxi Xinyanghe Biotechnology Co., Ltd.

[0025] Example 1 Taking the preparation of 1 L of immune adjuvant as an example, the immune adjuvant for preparing rabbit anti-CRP in this example includes: aluminum hydroxide 3.5 g / L, kaempferitrin 1.5 g / L, dodecylpyranoside 0.3 g / L, cyclodextrin-encapsulated ellagic acid 2 g / L, and the balance is deionized water.

[0026] The preparation method of the immune adjuvant in this example includes the following steps: S1, prepare the kaempferitrin solution Kaempferitrin is not easily soluble. It is necessary to prepare the solution first and then mix it with other reagents. Preparation of the kaempferitrin solution: Weigh 1.5 g of kaempferitrin, add 50 mL of deionized water, heat to 60 °C and stir until completely dissolved, and then cool to obtain the kaempferitrin solution.

[0027] S2, preparation of cyclodextrin-encapsulated ellagic acid Dissolve 15 g of hydroxypropyl-β-cyclodextrin in 60 mL of 0.1 M PBS buffer solution with a pH of 8.0, then add 3 g of ellagic acid, and continue to stir at 45 °C until a clear solution is formed. Subsequently, freeze-dry (pre-freeze at 50 °C for 6 h; primary drying at 20 Pa and -15 °C for 16 h; secondary drying at 25 °C for 10 h) to obtain the cyclodextrin-encapsulated ellagic acid powder.

[0028] S3, add 0.3 g of dodecylpyranoside to 700 mL of deionized water and stir to dissolve it. Then add 3.5 g of aluminum hydroxide and stir until dissolved. Then, under stirring, add the kaempferitrin solution in step S1. After dissolution, add 2 g of cyclodextrin-encapsulated ellagic acid, add deionized water to make up to 1 L, and stir until dissolved to obtain the immune adjuvant for rabbit anti-CRP.

[0029] Example 2 Taking the preparation of 1 L of immunoadjuvant as an example, the immunoadjuvant for preparing rabbit anti-CRP in this example includes: 3.5 g / L of aluminum hydroxide, 1.5 g / L of MPEG-NH2 modified kaempferide, 0.3 g / L of dodecyl glucopyranoside, 2 g / L of cyclodextrin-encapsulated ellagic acid, and the balance is deionized water.

[0030] The preparation method of the immunoadjuvant in this example includes the following steps: S1, Preparation of MPEG-NH2 modified kaempferide S11, Dissolve 3 g of kaempferide in 60 mL of organic solvent DMF, then add 4 g of EDC and 3 g of NHS, and adjust the pH to 7.5, stir and react for 25 min to obtain an activated kaempferide solution; S12, Dissolve 4.5 g of MPEG-NH2 in 80 mL of organic solvent DMF, and then slowly drop the activated kaempferide solution in step S11 into the MPEG-NH2 solution, stir while adding at 35 °C, stir for 10 h, maintain the pH at 7.5, and after the reaction is completed, rotary evaporate to remove the organic solvent to obtain MPEG-NH2 modified kaempferide.

[0031] S2, Preparation of cyclodextrin-encapsulated ellagic acid Dissolve 15 g of hydroxypropyl-β-cyclodextrin in 100 mL of 0.1 M PBS buffer solution with pH 8.0, then add 3 g of ellagic acid, continue to stir at 45 °C until a clear solution is formed, and then freeze-dry (pre-freeze at 50 °C for 6 h; primary drying at 20 Pa and -15 °C for 16 h; secondary drying at 25 °C for 10 h) to obtain cyclodextrin-encapsulated ellagic acid powder.

[0032] S3, Add 0.3 g of dodecyl glucopyranoside to 600 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 kaempferide under stirring, add 3 g of cyclodextrin-encapsulated ellagic acid after dissolution, add deionized water to make up to 1 L, and stir until dissolved to obtain the immunoadjuvant for rabbit anti-CRP.

[0033] Example 3 Taking the preparation of 1 L of immunoadjuvant as an example, the immunoadjuvant for preparing rabbit anti-CRP in this example includes: 1 g / L of aluminum phosphate, 2 g / L of MPEG-NH2 modified kaempferide, 0.8 g / L of dodecyl glucopyranoside, 3 g / L of cyclodextrin-encapsulated ellagic acid, and the balance is deionized water.

[0034] The preparation method of the immunoadjuvant in this example includes the following steps: S1, Preparation of MPEG-NH2 modified kaempferide S11. Dissolve 3 g of kaempferitrin in 60 mL of the organic solvent DMSO. Subsequently, add 3.6 g of EDC and 3.6 g of NHS, and adjust the pH to 8.5. Stir and react for 20 min to obtain an activated kaempferitrin solution. S12. Dissolve 3.6 g of MPEG-NH2 in 80 mL of the organic solvent DMSO. Then, slowly add the activated kaempferitrin solution obtained in step S11 to the MPEG-NH2 solution. At 30 °C, add while stirring, stir for 12 h, maintain the pH at 8.0. After the reaction, rotary evaporate to remove the organic solvent to obtain MPEG-NH2 modified kaempferitrin.

[0035] S2. Preparation of cyclodextrin inclusion ellagic acid Dissolve 18 g of hydroxypropyl-β-cyclodextrin in 180 mL of PBS buffer solution with pH 8.0. Then, add 3 g of ellagic acid and continue to stir at 50 °C until a clear solution is formed. Subsequently, freeze-dry (pre-freeze at 50 °C for 6 h; primary drying at 20 Pa and -15 °C for 16 h; secondary drying at 25 °C for 10 h) to obtain cyclodextrin inclusion ellagic acid powder.

[0036] S3. Add 0.8 g of dodecylpyranoside to 700 mL of deionized water and stir to dissolve it. Subsequently, add 1 g of aluminum phosphate and stir until dissolved. Then, add 2 g of MPEG-NH2 modified kaempferitrin under stirring. After dissolution, add 3 g of cyclodextrin inclusion ellagic acid, add deionized water to make up the volume to 1 L, and stir until dissolved to obtain an immune adjuvant for rabbit anti-CRP.

[0037] Example 4 Taking the preparation of 1 L of immune adjuvant as an example, the immune adjuvant for rabbit anti-CRP prepared in this example includes: aluminum phosphate 4 g / L, MPEG-NH2 modified kaempferitrin 0.5 g / L, dodecylpyranoside 1 g / L, cyclodextrin inclusion ellagic acid 2.5 g / L, and the balance is deionized water.

[0038] The preparation method of the immune adjuvant in this example includes the following steps: S1. Preparation of MPEG-NH2 modified kaempferitrin S11. Dissolve 3 g of kaempferitrin in 60 mL of the organic solvent DMF. Subsequently, add 3.5 g of EDC and 3.3 g of NHS, and adjust the pH to 8.5. Stir and react for 30 min to obtain an activated kaempferitrin solution. S12. Dissolve 6 g of MPEG-NH2 in 80 mL of the organic solvent DMF. Then, slowly add the activated kaempferitrin solution obtained in step S11 to the MPEG-NH2 solution. At 40 °C, add while stirring, stir for 6 h, maintain the pH at 8.0. After the reaction, rotary evaporate to remove the organic solvent to obtain MPEG-NH2 modified kaempferitrin.

[0039] S2, Preparation of Cyclodextrin-Encapsulated Ellagic Acid Dissolve 16 g of hydroxypropyl-β-cyclodextrin in 160 mL of 0.1 M PBS buffer at pH 8.0, then add 3 g of ellagic acid, and continue stirring at 40 °C until a clear solution is formed. Subsequently, freeze-dry (pre-freeze at 50 °C for 6 h; primary drying at 20 Pa and -15 °C for 16 h; secondary drying at 25 °C for 10 h) to obtain cyclodextrin-encapsulated ellagic acid powder.

[0040] S3, Add 1 g of dodecylpyranoside to 700 mL of deionized water and stir to dissolve it. Subsequently, add 4 g of aluminum phosphate and stir until dissolved. Then, slowly dropwise add 0.5 g of MPEG-NH2-modified kaempferol while stirring. After dissolution, add 2.5 g of cyclodextrin-encapsulated ellagic acid, and make up the volume to 1 L with deionized water, and stir until dissolved to obtain an adjuvant for rabbit anti-CRP.

[0041] Example 5 Taking the preparation of 1 L of adjuvant as an example, the adjuvant for rabbit anti-CRP prepared in this example includes: 5 g / L of aluminum hydroxide, 3 g / L of MPEG-NH2-modified kaempferol, 0.1 g / L of dodecylpyranoside, 1 g / L of cyclodextrin-encapsulated ellagic acid, and the balance is deionized water.

[0042] The preparation method of the adjuvant in this example includes the following steps: S1, Preparation of MPEG-NH2-Modified Kaempferol S11, Dissolve 3 g of kaempferol in 60 mL of organic solvent DMF, then add 4.5 g of EDC and 3.5 g of NHS, and adjust the pH to 8, and stir and react for 20 min to obtain an activated kaempferol solution; S12, Dissolve 5 g of MPEG-NH2 in 60 - 100 mL of organic solvent DMF or DMSO, and then slowly dropwise add the activated kaempferol solution in step S11 to the MPEG-NH2 solution. At 35 °C, add while stirring, stir for 8 h, maintain the pH at 7.5, and after the reaction, rotary evaporate to remove the organic solvent to obtain MPEG-NH2-modified kaempferol.

[0043] S2, Preparation of Cyclodextrin-Encapsulated Ellagic Acid Dissolve 17 g of hydroxypropyl-β-cyclodextrin in 170 mL of 0.1 M PBS buffer at pH 7.5, then add 3 g of ellagic acid, and continue stirring at 50 °C until a clear solution is formed. Subsequently, freeze-dry (pre-freeze at 50 °C for 6 h; primary drying at 20 Pa and -15 °C for 16 h; secondary drying at 25 °C for 10 h) to obtain cyclodextrin-encapsulated ellagic acid powder.

[0044] S3. Add 0.1 g of dodecyl glucopyranoside to deionized water and stir to dissolve it. Then add 5 g of aluminum hydroxide and stir until dissolved. Next, add 3 g of MPEG-NH2 modified kaempferol glycoside under stirring. After dissolution, add 1 g of cyclodextrin to complex ellagic acid, and add deionized water to make up to 1 L. Stir until dissolved to obtain the adjuvant for rabbit anti-CRP.

[0045] Comparative Example 1 Compared with Example 1, the adjuvant in Comparative Example 1 lacks kaempferol glycoside, and the rest is the same as in Example 1.

[0046] Comparative Example 2 Compared with Example 1, the adjuvant in Comparative Example 2 lacks cyclodextrin-complexed ellagic acid, and the rest is the same as in Example 1.

[0047] Comparative Example 3 Compared with Example 1, the adjuvant in Comparative Example 3 lacks dodecyl glucopyranoside, and the rest is the same as in Example 1.

[0048] Comparative Example 4 Compared with Example 1, the concentration of kaempferol glycoside in the adjuvant in Comparative Example 4 exceeds the defined range, specifically 5 g / L, and the rest is the same as in Example 1.

[0049] Comparative Example 5 Compared with Example 1, the concentration of cyclodextrin-complexed ellagic acid in the adjuvant in Comparative Example 5 exceeds the defined range, specifically 5 g / L, and the rest is the same as in Example 1.

[0050] Comparative Example 6 Compared with Example 2, in MPEG-NH2 modified kaempferol glycoside in Comparative Example 6, the dosage of MPEG-NH2 is 2.5 times the weight of kaempferol glycoside, and the rest is the same as in Example 2.

[0051] Immune potency detection Use the adjuvants prepared in the above examples and comparative examples to immunize rabbits, with 3 rabbits for each example or comparative example, and verify their immune effects.

[0052] (1) The antigen and animal information selected for the immune experiment are as follows: The antigen is C Reactive Protein, with a concentration of 2.47 mg / mL (OD at 280 nm), sourced from Human, and purchased from ADVY CHEMICAL PVT.LTD. Antigen preservation system: 50 mM Tris at pH 8.0 containing 200 mM NaCl, 5 mM CaCl and 15 mM NaN3.

[0053] Dilute the CRP antigen to a concentration of 1.2 mg / mL with 0.01M PBS for later use.

[0054] Animals: Japanese white rabbits with big ears, 8 - 12 weeks old, female, purchased from Wuhan Wanqian Jiaxing Biotechnology Co., Ltd.

[0055] (2)Immunization protocol: 1. First immunization: Mix 100 μg of antigen with 100 μg of immune adjuvant (mass ratio 1:1), fix the rabbit, and inject subcutaneously at the back of the neck to immunize the animal. 2. Second immunization: On the 17th day after the first immunization, use the same amount of antigen and immune adjuvant as in the first immunization, and inject subcutaneously at the back of the neck to immunize the animal. 3. Collect blood from the animal on the 28th day and detect the titer by ELISA.

[0056] (3)Establishment of serum titer determination by ELISA method: 1. Antigen coating: Coat the CRP antigen (2 μg / mL) in the enzyme - linked immunosorbent assay (ELISA) plate and incubate overnight at 4°C.

[0057] 2. Blocking: Add the blocking solution (2% BSA), incubate at 37°C for 2 h, then discard the blocking solution and pat dry, and dry at 37°C for 2 h.

[0058] 3. Add the serum to be tested: Dilute the serum to be tested starting from 1:1000, and then perform a two - fold serial dilution in each well. Incubate at 37°C for 30 min, wash the plate (PBST) and pat dry.

[0059] 4. Add the 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 min, wash the plate and pat dry.

[0060] 5. Color development: Add the chromogenic solution (TMB), react at 37°C for 10 min, and measure the OD value with a microplate reader at dual wavelengths.

[0061] Table 1 Absorbance values of the examples and comparative examples

[0062] As shown in Table 1, the immune adjuvants in the examples of the present invention are all higher than those in the comparative examples, specifically higher than 50W. This proves that the immune adjuvant of the present invention has the effect of increasing the immune titer. From Examples 1 and 2, it can be seen that after the modification of kaempferol by MPEG - NH2, the immune titer has been improved to a certain extent. This is because after the modification of kaempferol by MPEG - NH2, on the one hand, the solubility of kaempferol is increased, improving the immune - stimulating efficacy of kaempferol, and on the other hand, the synergistic effect between the components is also increased.

[0063] As can be seen from Comparative Examples 1-3, the lack of any one of kaempferitrin, dodecyl glucopyranoside, and cyclodextrin-encapsulated ellagic acid will reduce the immune titer, thus proving that the components have an immune-enhancing effect under their synergistic action.

[0064] As can be seen from Comparative Examples 4-5, when the dosages of kaempferitrin and cyclodextrin-encapsulated ellagic acid exceed the defined range, the immune titer does not increase but decreases. This is because some immune pathways may produce negative feedback regulation due to overactivation, inhibiting the immune titer; in addition, after exceeding the range, the synergistic effect is disrupted, resulting in a decrease in the immune effect.

[0065] As can be seen from Comparative Example 6, when the dosage of MPEG-NH2 exceeds the defined range, the immune titer is reduced instead. This may be because excessive MPEG-NH2 leads to too long or too dense molecular chains, forming steric hindrance and preventing the effective binding of kaempferitrin to the surface receptors of immune cells.

[0066] The second immunization in Example 1 was set on the 16th and 18th days. The serum titer was detected and measured according to the above method, and the results are shown in Table 2.

[0067] Table 2 Effects of the second immunization time on the serum titer

[0068] As can be seen from Table 2, when immunizing on the 16th - 18th days, there was no significant difference in the serum titer, and it could reach about 500,000 in both cases. In actual operation, any day between the 16th - 18th days can be selected for the second immunization.

[0069] II. Detection of stimulation index To analyze the immune effect of the immune adjuvant, the following experiment was carried out in the present invention: After the immunization ended, the experimental rabbits were sacrificed, and spleen cells were taken to prepare a spleen cell suspension, ensuring that the proportion of viable cells ≥ 95%, and diluting the spleen cell suspension to 1×10 7 cells / mL. 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 solution (5 μg / mL), 100 μL of pokeweed (5 μg / mL), and 100 μL of phytohemagglutinin (5 μg / mL) were added respectively, with 5 wells in each group. The control group was added with 100 μL of 1640 culture medium. Incubate at 37°C and 5% CO2 for 68 h. Subsequently, the MTT method was used to detect cell proliferation, and the stimulation index (SI) was calculated as the OD value of the mitogen culture / the OD value of the non-mitogen culture. The results are shown in Table 3.

[0070] Table 3 Stimulation indexes of Examples and Comparative Examples

[0071] As can be seen from Table 3, compared with the comparative examples, the immunoadjuvant of the embodiment of the present invention can significantly enhance the proliferation response of CRP-immunized rabbit spleen cells induced by three mitogens, namely ConA, PWM, and PHA.

[0072] As can be seen from Comparative Examples 1-3, the synergistic effect of kaempferol, dodecyl glucopyranoside, and cyclodextrin-encapsulated ellagic acid enhances the cellular immune response intensity of experimental rabbits to immunogens. The lack of any one of these components will weaken the cellular immune response intensity, which is similar to the results in Table 1.

[0073] As can be seen from Comparative Examples 4-6, when the dosage of each component exceeds the defined range, negative feedback regulation will occur, inhibiting the immune response and thus leading to a decrease in the immune response intensity.

[0074] 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 principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An immune adjuvant for preparing rabbit anti-CRP, characterized in that: It includes the following components: aluminum salt, kaempferitrin, dodecyl glucopyranoside, cyclodextrin-encapsulated ellagic acid, and deionized water.

2. The immune adjuvant for preparing rabbit anti-CRP according to claim 1, characterized in that: Based on a mass concentration of 100%, the mass concentrations of the components in the immunoadjuvant are: aluminum salt 0.1% - 0.5%, kaempferitrin 0.05% - 0.3%, dodecyl glucopyranoside 0.01% - 0.1%, cyclodextrin-encapsulated ellagic acid 0.1% - 0.3%, and the balance is deionized water.

3. An immune adjuvant for preparing rabbit anti-CRP according to claim 1, characterized in that: The method for cyclodextrin-encapsulating ellagic acid is as follows: Dissolve hydroxypropyl-β-cyclodextrin in PBS buffer solution with a pH of 7.5 - 8.0, then add ellagic acid, and continue stirring at 40 - 50 °C until a clear solution is formed. Subsequently, freeze-dry to obtain cyclodextrin-encapsulated ellagic acid powder.

4. An immune adjuvant for preparing rabbit anti-CRP according to claim 3, characterized in that: The mass ratio of the hydroxypropyl-β-cyclodextrin to ellagic acid is 5 - 6:

1.

5. An immune adjuvant for preparing rabbit anti-CRP according to claim 1, characterized in that: The kaempferitrin is chemically modified with MPEG-NH2, and the modification method includes the following steps: S1. Dissolve kaempferitrin in an organic solvent, then add EDC and NHS, and adjust the pH to 7.5 - 8.

5. Stir and react for 20 - 30 min to obtain an activated kaempferitrin solution. S2. Dissolve MPEG-NH2 in an organic solvent, then slowly drop the activated kaempferitrin solution into the MPEG-NH2 solution, stir while adding at 30 - 40 °C, stir for 6 - 12 h, maintain the pH at 7.5 - 8.

0. After the reaction, rotary evaporate to remove the organic solvent to obtain MPEG-NH2-modified kaempferitrin.

6. An immune adjuvant for preparing rabbit anti-CRP according to claim 5, characterized in that: In step S1, the mass ratio of the kaempferitrin:EDC:NHS is 1:1.2 - 1.5:1 - 1.

2.

7. An immune adjuvant for preparing rabbit anti-CRP according to claim 5, characterized in that: In step S2, the mass ratio of the MPEG-NH2 to kaempferitrin is 1.2 - 2:

1.

8. An immune adjuvant for preparing rabbit anti-CRP according to claim 5, characterized in that: The aluminum salt is one of aluminum hydroxide, aluminum phosphate, and aluminum sulfate; the organic solvent is DMF or DMSO.

9. A method for preparing an immune adjuvant for preparing rabbit anti-CRP according to any one of claims 5-8, characterized in that: It includes the following steps: Add dodecyl glucopyranoside to deionized water and stir to dissolve it. Subsequently, add the aluminum salt and stir until dissolved. Then, add MPEG-NH2-modified kaempferitrin under stirring, and after dissolution, add cyclodextrin-encapsulated ellagic acid and stir until dissolved to obtain the immunoadjuvant for rabbit anti-CRP.

10. A method for immunizing an animal with the immunoadjuvant according to any one of claims 1-8, characterized in that: It includes the following steps: S1. First immunization: Mix the antigen and the immunoadjuvant, and immunize the animal by subcutaneous injection at the nape of the neck. S2. Second immunization: On the 16th - 18th day after the first immunization, use the same dosage of the antigen and the immunoadjuvant as in the first immunization, and immunize the animal by subcutaneous injection at the nape of the neck. S3. Collect blood from the animal on the 28th day to detect the titer. The antigen and the immunoadjuvant are mixed in equal volume or equal mass.

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