A water-soluble rapid immune adjuvant and its preparation method

Abstract

The present invention provides a water-soluble rapid immune adjuvant, which comprises 7α,25-OHC, cyclodextrin, aluminum salt, chitosan-based thermosensitive hydrogel and deionized water. When the water-soluble rapid immune adjuvant of the present invention is used, emulsification is not required. After the antigen and the adjuvant are mixed evenly, they are directly injected for immunization in a water-soluble manner, which is convenient to operate and avoids the destruction of the antigen during the emulsification process. Moreover, it will not cause the eversion of hydrophobic groups, retains the natural conformation of the antigen, and can obtain antibodies against conformational epitopes.

Description

Technical Field

[0001] The present invention relates to the technical field of immune adjuvants, and particularly to a water-soluble rapid immune adjuvant and a preparation method thereof. Background Art

[0002] With the rapid development of immunology, people have developed a variety of immunological applications based on antigen-antibody reactions, including the development and use of vaccines, the clinical application of therapeutic antibody drugs, the production of scientific research raw materials and in vitro diagnostic kits, etc. The development and production of high-affinity, high-titer, and high-specificity antibodies are essential for all these applications. Whether it is traditional monoclonal and polyclonal antibody development or the newly developed library display technology in recent years, antigen immunization of animals is inevitable. Therefore, the key technical link for the successful implementation of various applications is the ability to immunize animals successfully and efficiently. Thanks to the development of purification chromatography technology, people can usually obtain highly pure and biologically active antigens. However, immunization with antigens alone often fails to induce an effective immune response in the body. An effective way to solve this key problem is to use adjuvants.

[0003] Traditional animal immunization uses the method of emulsifying antigens with classical Freund's adjuvant and then injecting them into animals. However, this method has some problems: the mixing of antigens and oily adjuvants requires emulsification, and the natural conformation of antigens is inevitably damaged during emulsification, 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 immune effects; the BCG component in complete adjuvant belongs to a controlled substance and is difficult to obtain, and the complex protein components increase the immune background; Freund's complete adjuvant may cause local inflammation and other diseases at the injection site. New adjuvants that have emerged on the market have made certain improvements to the safety issues of adjuvants, such as using squalene that is easily degraded and metabolized by the body for emulsification, using small molecule substances to replace the role of BCG, or using glycoside and lipid complexes to enhance antigen presentation. However, these adjuvants often have different applicability for antigens of different properties, with large fluctuations in immune effects and inability to effectively maintain the antigen structure. In addition, most adjuvants are ineffective for immunization of antigens with high homology or low immunogenicity. Summary of the Invention

[0004] In view of this, the present invention provides a water-soluble rapid immune adjuvant that does not require emulsification, does not contain complex protein components such as interleukin or inactivated mycobacteria, and does not produce antibodies against the adjuvant after immunization, as well as a preparation method thereof.

[0005] The technical solution of the present invention is realized as follows: The present invention provides a water-soluble rapid immune adjuvant, characterized in that it includes 7α,25-OHC, cyclodextrin, aluminum salt, chitosan-based thermosensitive hydrogel, and deionized water.

[0006] Based on the above technical solutions, preferably, taking the preparation of 1 L of adjuvant as an example, it includes 50 - 500 μg / mL of 7α,25-OHC, 1% - 3% of cyclodextrin, 1 - 5 mg / mL of aluminum salt, 5% - 7% of chitosan-based thermosensitive hydrogel, and the rest is deionized water.

[0007] Based on the above technical solutions, preferably, the chitosan-based thermosensitive hydrogel is conjugated with imidazoquinoline.

[0008] Based on the above technical solutions, preferably, the imidazoquinoline is R848.

[0009] Based on the above technical solutions, preferably, the preparation method of the chitosan-R848 includes the following steps:

[0010] S1, Add chitosan powder into deionized water, disperse it evenly by ultrasonic wave, then dropwise add lactic acid while stirring until the chitosan is completely dissolved, add bromoacetic acid to adjust the pH to 7 - 7.5, and react at 20 - 30 °C for 2 h to obtain carboxylated chitosan;

[0011] S2, Add EDC, NHS, and R848 into the carboxylated chitosan in sequence, react at 20 - 30 °C for 1 - 1.5 h, then place the reaction solution in an environment of 4 °C, add an aqueous solution of β-glycerophosphate with a mass concentration of 10.8%, and mix evenly to obtain a chitosan-based thermosensitive hydrogel conjugated with R848.

[0012] Based on the above technical solutions, preferably, the deacetylation degree of the chitosan powder is 75% - 99%, the molecular weight is 10 kd - 500 kd, the viscosity is 30 - 100 cP, and the mass ratio of the chitosan to deionized water is (1 - 2):100.

[0013] Based on the above technical solutions, preferably, the molar ratio of the chitosan to bromoacetic acid in step S1 is (1 - 3):20.

[0014] Based on the above technical solutions, preferably, the molar ratio of the carboxylated chitosan:EDC:NHS:R848 in step S2 is 500:(1 - 2):(1 - 3):25.

[0015] Based on the above technical solutions, preferably, the aluminum salt is aluminum hydroxide.

[0016] The present invention also provides a preparation method of a water-soluble rapid immune adjuvant, including the following steps: Dissolve 7α,25-OHC, cyclodextrin, aluminum salt, and chitosan-based thermosensitive hydrogel in deionized water, and sterilize it by steam at 1.23×10 5 Pa for 30 min to obtain it.

[0017] In the water-soluble rapid immune adjuvant of the present invention, 7α,25-OHC promotes the migration of lymphocytes to the antigen injection site as a chemokine, cyclodextrin serves as a stabilizer for this immune adjuvant, and aluminum salts promote antigen presentation; chitosan-R848 is a macromolecule formed by coupling chitosan and R848 through amino groups, which can play a role in sustained TLR activation by fixing R848 at the antigen injection site while also acting as a slow-release adjuvant for other components and antigens. It has the following beneficial effects compared with the prior art:

[0018] (1) The water-soluble rapid immune adjuvant of the present invention does not contain components with complex proteins such as interleukin or inactivated mycobacteria, and no antibodies against the adjuvant are produced after immunization. At the same time, since it does not contain mineral oil, the adjuvant components of the present invention are all water-soluble and biodegradable absorbable substances, with little tissue damage and stress response.

[0019] (2) The water-soluble rapid immune adjuvant of the present invention is a water-soluble adjuvant that does not cause the eversion of hydrophobic groups and retains the natural conformation of the antigen. Therefore, antibodies against conformational epitopes can be obtained. When using this water-soluble rapid immune adjuvant to mix antigens for immunization, emulsification is not required. After the antigen and adjuvant are mixed evenly, they are directly injected for immunization in a water-soluble manner. This not only makes the operation convenient but also avoids the destruction of the antigen during the emulsification process, and it is easier to obtain antibodies against structural epitopes. Compared with traditional Freund's adjuvant and new adjuvants on the market, it can produce higher antibody serum titers.

[0020] (3) The water-soluble rapid immune adjuvant of the present invention can break animal immune tolerance, and high-titer and high-affinity antibodies can still be prepared for antigens with high homology. Only 2 immunizations are required to prepare antibodies using the water-soluble rapid immune adjuvant of the present invention. The number of immunization needles is small, and the antigen dosage per needle is as low as 10 - 20 μg for mice and 50 - 100 μg for rabbits, effectively reducing the antigen usage. Antibodies are produced quickly, and antibodies can be successfully obtained in 3 - 5 weeks, saving half of the time compared with conventional Freund's adjuvant, greatly improving the experimental efficiency and saving the animal feeding cost. 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 use in 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, other drawings can be obtained based on these drawings without creative efforts.

[0022] Figure 1 It is a detection graph of the immune effects of each group in the screening of the components of the water-soluble rapid immune adjuvant of the present invention;

[0023] Figure 2Figure showing the effects of different immune adjuvants on cellular immunity in OVA-immunized mice;

[0024] Figure 3 Figure showing the effects of different immune adjuvants on humoral immunity in OVA-immunized mice. Detailed implementation manners

[0025] The following will describe the technical solutions in the embodiments of the present invention clearly and completely in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0026] 7α,25-OHC and R848 of the present invention are purchased from MedChemExpress, a supplier of bioactive compounds; the cyclodextrin is β-cyclodextrin and is purchased from sigma.

[0027] Example 1

[0028] Taking the preparation of 1 L of adjuvant as an example, the water-soluble rapid immune adjuvant in Example 1 includes 50 μg / mL of 7α,25-OHC, 1% of β-cyclodextrin, 1 mg / mL of aluminum hydroxide, and 5% of chitosan-based thermosensitive hydrogel conjugated with R848, and the rest is deionized water.

[0029] The preparation method of the chitosan-based thermosensitive hydrogel conjugated with R848 includes the following steps:

[0030] S1. Add low-viscosity chitosan powder with a deacetylation degree of 75%, a molecular weight of 10 kd, and a viscosity of 30 to deionized water. The mass concentration of chitosan is 1%. Ultrasonically disperse it evenly, then dropwise add lactic acid while stirring until the chitosan is completely dissolved. Add bromoacetic acid to adjust the pH to 7 and react at 20 °C for 2 h to obtain carboxylated chitosan; the molar ratio of chitosan to bromoacetic acid is 1:20;

[0031] S2. Sequentially add EDC, NHS, and R848 to the carboxylated chitosan. The molar ratio of carboxylated chitosan:EDC:NHS:R848 is 500:1:1:25; react at 20 °C for 1 h, then place the reaction solution in a 4 °C environment, add an aqueous solution of β-glycerophosphate with a mass concentration of 10.8%, and mix evenly to obtain the chitosan-based thermosensitive hydrogel conjugated with R848.

[0032] Example 2

[0033] For the water-soluble rapid immune adjuvant of Example 2, taking the preparation of 1 L of adjuvant as an example, it includes 7α,25-OHC at 200 μg / mL, β-cyclodextrin at 2%, aluminum hydroxide at 3 mg / mL, and chitosan-based thermosensitive hydrogel conjugated with R848 at 6%, and the rest is deionized water.

[0034] The preparation method of the chitosan-based thermosensitive hydrogel conjugated with R848 includes the following steps:

[0035] S1. Add low-viscosity chitosan powder with a deacetylation degree of 85%, a molecular weight of 350 kd, and a viscosity of 60 cP into deionized water. The mass concentration of chitosan is 1.5%. Disperse it evenly by ultrasonic wave, then dropwise add lactic acid while stirring until the chitosan is completely dissolved. Add bromoacetic acid to adjust the pH to 7.3, and react at 25 °C for 2 h to obtain carboxylated chitosan; the molar ratio of chitosan to bromoacetic acid is 3:20.

[0036] S2. Sequentially add EDC, NHS, and R848 into the carboxylated chitosan. The molar ratio of carboxylated chitosan: EDC: NHS: R848 is 500:2:3:25. React at 25 °C for 1.2 h, then place the reaction solution in a 4 °C environment, add an aqueous solution of β-glycerophosphate sodium with a mass concentration of 10.8%, and mix evenly to obtain the chitosan-based thermosensitive hydrogel conjugated with R848.

[0037] Example 3

[0038] For the water-soluble rapid immune adjuvant of Example 3, taking the preparation of 1 L of adjuvant as an example, it includes 7α,25-OHC at 500 μg / mL, β-cyclodextrin at 3%, aluminum hydroxide at 5 mg / mL, and chitosan-based thermosensitive hydrogel conjugated with R848 at 7%, and the rest is deionized water.

[0039] The preparation method of the chitosan-based thermosensitive hydrogel conjugated with R848 includes the following steps:

[0040] S1. Add low-viscosity chitosan powder with a deacetylation degree of 99%, a molecular weight of 500 kd, and a viscosity of 100 cP into deionized water. The mass concentration of chitosan is 2%. Disperse it evenly by ultrasonic wave, then dropwise add lactic acid while stirring until the chitosan is completely dissolved. Add bromoacetic acid to adjust the pH to 7.5, and react at 30 °C for 2 h to obtain carboxylated chitosan; the molar ratio of chitosan to bromoacetic acid is 1:10.

[0041] S2. Add EDC, NHS, and R848 to carboxylated chitosan in sequence. The molar ratio of carboxylated chitosan:EDC:NHS:R848 is 500:1.5:2:25. React at 30 °C for 1.5 h, then place the reaction solution in an environment at 4 °C, add an aqueous solution of β-glycerophosphate sodium with a mass concentration of 10.8%, and mix evenly to obtain a chitosan-based thermosensitive hydrogel conjugated with R848.

[0042] The preparation methods of the water-soluble rapid immune adjuvants in Examples 1 to 3 are as follows: Dissolve 7α,25-OHC, cyclodextrin, aluminum salt, and chitosan-based thermosensitive hydrogel in deionized water, and sterilize by steam at 1.23×10 5 Pa for 30 min to obtain the product.

[0043] Screening of each component of the water-soluble rapid immune adjuvant: Select 15 healthy male Balb / c mice at 6 - 8 weeks old, divide them into 5 groups with 3 mice in each group. Prepare water-soluble rapid immune adjuvants by combining some or all of 7α,25-OHC, cyclodextrin, aluminum salt, and chitosan-R848 (chitosan-based thermosensitive hydrogel conjugated with R848), and set up the experimental groups as follows:

[0044] Table 1 Formulas of experimental groups for immune adjuvant screening

[0045]

[0046] Mix 50 μL of various immune adjuvants with 50 μL of the prepared antigen, immunize each group of mice subcutaneously in the nape of the neck twice. The interval between the first immunization and the second immunization is 3 weeks. Sacrifice the mice 2 weeks after the second immunization and separate the serum. At the same time, aseptically take the spleen to prepare a spleen cell suspension. Use a T lymphocyte subset kit to detect the T lymphocyte subsets of peripheral blood lymphocytes and the spleen, and calculate the ratio of lymphocyte subsets CD4 + / CD8 + (mean ± standard deviation).

[0047] The experimental results are shown in Table 1. Among them, the ratios of CD4 + / CD8 + of peripheral blood and spleen lymphocytes in experimental groups C, D, and E after immunization are close, and are all significantly lower than those in experimental groups A and B. Select the components corresponding to experimental group A with the highest ratio of CD4 + / CD8 + as the components of the water-soluble rapid immune adjuvant.

[0048] In addition, for the ELISA detection of serum titer, the antigen OVA at a concentration of 1 μg / mL was coated and incubated overnight at 4°C. The microtiter plate was blocked with 2% BSA and incubated at 37°C for 2 h. The plate was washed twice with PBST, and then incubated with the antiserum (serially diluted from 1:500) at 37°C for 1 h. After washing the plate three times with PBST, it was incubated with HRP-labeled goat anti-mouse IgG (diluted 1:5000) at 37°C for 45 min. After washing the plate, TMB was added for color development, and the OD value was read using an ELISA reader.

[0049] The experimental results are shown in Figure 1 , where the vertical axis represents the absorbance value of the ELISA-detected samples, and the horizontal axis represents the different dilution ratios of the antiserum after mouse immunization. Curves A to E are the curves plotted from the average ELISA detection values of the diluted mouse sera in experimental groups A, B, C, D, and E, respectively. Experimental group A contains all the components of the adjuvant described in the patent, and its antiserum titer is also the highest among all experimental groups. Experimental groups B, C, and D contain only partial components of the adjuvant described in the patent. Among them, the antiserum titer of group B is also significantly higher than that of group E immunized with Freund's adjuvant. It can be seen that the components of the adjuvant described in the present invention have a synergistic effect in enhancing immunity.

[0050] Table 1. Detection of lymphocyte subsets

[0051]

[0052] I. Effects of the water-soluble rapid immune adjuvant on the cellular immunity of OVA-immunized mice

[0053] Fifteen healthy male Balb / c mice aged 6 - 8 weeks were randomly divided into 5 groups of 3 mice each. The following 5 experimental groups were set up:

[0054] Table 2 Formulation of experimental groups

[0055]

[0056] Each group was immunized subcutaneously in the neck and back twice, with a 3-week interval between the first and second immunizations. Two weeks after the second immunization, the mice were bled to death, and the spleens were aseptically removed, ground, filtered, added with Hank's solution, centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and the washing was repeated twice. The cells were resuspended in 1640 medium. Take 0.1 mL of the spleen cell suspension for cell counting, and the number of viable cells was not less than 95%. According to the counting results, the spleen cell suspension was diluted to 1×107 cells / mL, and 100 μL was added to a 96-well plate. The spleen cell suspension of each mouse was replicated and plated in 12 wells. Among them, 100 μL of concanavalin A solution (ConA) (5 μg / mL) was added to 3 wells, 100 μL of pokeweed mitogen (PWM) (5 μg / mL) was added to 3 wells, 100 μL of phytohemagglutinin (PHA) (5 μg / mL) was added to 3 wells, and 100 μL of 1640 culture medium was added to 3 wells. Incubate at 37°C and 5% CO2 for 68 h. Four hours before the end, 50 μL of MTT solution (2 mg / mL) was added to each well and incubated for another 4 h. Discard the liquid in each well, add 150 μL of solution DMSO: 1N HCl (9:1), place it in the dark at room temperature for 15 min, and measure the OD value at a wavelength of 578 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the stimulation index (SI) = OD value of the mitogen culture / OD value of the non-mitogen culture.

[0057] The experimental results are shown in Figure 2 , compared with the aluminum adjuvant control group and the Freund's adjuvant control group, the water-soluble rapid immune adjuvant test group could significantly enhance the proliferation response of splenocytes induced by ConA, PWM, and PHA in OVA-immunized mice. It can be seen that the water-soluble rapid immune adjuvant can significantly enhance the cellular immune response of OVA-immunized mice, and the enhancement effect is better than that of Freund's adjuvant, while the aluminum adjuvant alone has no such activity.

[0058] II. Effect of water-soluble rapid immune adjuvant on humoral immunity of OVA-immunized mice

[0059] Fifteen healthy male Balb / c mice aged 6-8 weeks were randomly divided into groups of 3 each. The following 5 test groups were set up:

[0060] Table 3 Formulation of test groups

[0061]

[0062] Each group was immunized subcutaneously in the neck and back twice, with a 3-week interval between the first and second immunizations. Two weeks after the second immunization, the serum was separated, and the serum titer of each group of mice was detected by the Elisa method in Example 1.

[0063] The experimental results are shown in Figure 3, compared with the aluminum adjuvant control group and Freund's adjuvant control group, the water-soluble rapid immune adjuvant test group can significantly enhance the antiserum titer of OVA-immunized mice. It can be seen that the water-soluble rapid immune adjuvant can significantly enhance the humoral immune response of OVA-immunized mice, and the effect is better than that of Freund's adjuvant and aluminum adjuvant.

[0064] III. Preparation of GAPDH mouse monoclonal antibody using water-soluble rapid immune adjuvant

[0065] The water-soluble rapid immune adjuvant of Example 1 of the present invention is used to prepare GAPDH mouse monoclonal antibody, and the preparation method includes the following steps:

[0066] 1. Immunization of experimental mice

[0067] 1.1 Select 4 healthy male Balb / c mice at 6-8 weeks old.

[0068] 1.2 The antigen GAPDH for injecting mice is 50 μL. It is more suitable to initially immunize 20 μg of antigen per mouse, and 10 μg is sufficient for booster immunization. When the volume is insufficient, it can be supplemented with PBS.

[0069] 1.3 Mix 50 μL of the water-soluble rapid immune adjuvant with the prepared 50 μL of antigen, gently pipette to mix well, and no emulsification is required.

[0070] 1.4 Each mouse is immunized at 2 sites (both the nape and thigh roots are acceptable), and 50 μL is subcutaneously injected at each site. After injection, wait for a few seconds to prevent antigen outflow.

[0071] 1.5 Booster immunization is carried out 3 weeks after the first immunization, and blood is collected 7-10 days after immunization (including blood collection for mid-term testing and final bleeding).

[0072] 2. Elisa titer detection

[0073] 2.1 Antigen coating: Add 100 μL of 1 μg / mL antigen GAPDH to each well of a 96-well plate, and incubate overnight at 4°C, or incubate at 37°C for 2 h.

[0074] 2.2 Blocking: Pour out the antigen solution, add 200 μL of blocking solution 2% BSA to each well, and incubate overnight at 4°C or incubate at 37°C for 2 h.

[0075] 2.3 Plate washing: Pour out the blocking solution, pat on the absorbent paper to try to suck out the residual liquid as much as possible, wash the plate three times with the washing solution, and try to pat dry the residual liquid each time. If it needs to be left for a period of time, dry it at 37°C, seal it with a sealed bag, and store it at -20°C.

[0076] 2.4 Incubation with primary antibody: Take the well-coated 96-well plate. Add 100 μL of negative serum reference solution to the first well, and add 1:500 antiserum to be tested to the second well. Subsequently, each subsequent well is serially diluted based on this. Incubate at 37 °C for 1 h.

[0077] 2.5 Incubation with secondary antibody: Pour out the liquid, wash the plate three times with the washing solution, and pat dry. Add 100 μL of HRP-labeled goat anti-mouse IgG (diluted 1:5000) to each well, and incubate at 37 °C for 45 min.

[0078] 2.6 TMB color development: Pour out the liquid, wash the plate three times with the washing solution, and pat dry. Add 100 μL of TMB substrate (self-made single-component TMB) to each well and incubate at 37 °C for 5 - 20 min (determine the color development time according to the color depth).

[0079] 2.7 Data analysis: Add 50 μL of stop solution (H2SO4) to each well, and read the absorbance value at a wavelength of 450 nm on the microplate reader. After the antibody titer detection reaches more than 20,000, the mice can be subjected to hybridoma fusion.

[0080] 3. Hybridoma fusion

[0081] 3.1 Preparation of immune spleen cells:

[0082] 1) Take the immunized Balb / c mice, draw blood from the eye socket to sacrifice them, immerse them in 75% ethanol for 5 min, and use forceps to hold them and stir them in 75% ethanol from time to time to fully disinfect them.

[0083] 2) Lay the sterilized filter paper on the fixing plate (with the inner side facing up), and take the mouse out of 75% alcohol and drain the alcohol.

[0084] 3) Use a sterile fixing needle to fix the mouse on its side (with the left side of the body facing up) on the fixing plate.

[0085] 4) Gently lift the abdominal skin of the mouse with sterile forceps, cut a slit from bottom to top with a sterile scissors, tear the skin of the mouse along the slit, and fix it.

[0086] 5) Put 3 - 5 mL of 1640 incomplete medium into the homogenizer. Use scissors and forceps to open the mouse's abdominal cavity, take out the spleen on the left side, cut off the surface fat, and put it into the homogenizer to gently grind.

[0087] 6) Filter the ground liquid through a cell sieve to remove lumps.

[0088] 7) Take 3 - 5 mL of 1640 incomplete medium to rinse the homogenizer and sieve it again.

[0089] 8) Pour the liquid filtered by the cell sieve into a clean 50mL sterile centrifuge tube, place it in a 50mL sterile centrifuge tube, add 1640 incomplete medium to 30mL, centrifuge at 1500rpm for 5min, discard the supernatant, resuspend the cell pellet with 1640 incomplete medium, and set aside.

[0090] 3.2 Cell fusion

[0091] 1) Resuspend SP2 / 0 cells and immune mouse spleen cells in 1640 incomplete medium, count them separately using a hemacytometer, mix them in a 50 mL sterile centrifuge tube at a ratio of spleen cells:SP2 / 0 cells = 1:3, mix thoroughly, and add 1640 incomplete medium to 40 mL.

[0092] 2) Centrifuge at 1500 rpm for 5 min. Prepare warm water at 37°C for incubation of the cell fusion centrifuge tube.

[0093] 3) After centrifugation, discard the supernatant and the liquid on the tube wall (it can be dried with sterile absorbent paper strips), gently tap the mixed precipitate of SP2 / 0 cells and immune mouse cells to loosen it, and immerse the bottom of the centrifuge tube in 37°C warm water.

[0094] 4) Take out 1 mL of the incubated fusion agent PEG1450 from the incubator and evenly drip it into the mixed cell pellet within 60 seconds (rotate the centrifuge tube while adding).

[0095] 5) Incubate for 45 seconds, take out the 1640 incomplete culture medium preheated in the 37°C incubator, take out 1 mL, and evenly drip it into the precipitate over 60 seconds to dilute the PEG fusion agent (rotate the centrifuge tube while adding), then take out another 1 mL and evenly drip it over 30 seconds, then slowly drip the remaining 45 mL of culture medium.

[0096] 6) Gently invert to mix, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0097] 7) Resuspend the cell pellet with 200 mL of feeder cell and HAT complete medium solution; if feeder cells have been plated in advance, only 100 mL of HAT complete medium solution is needed to resuspend. Dispense into 96-well cell culture plates, 100 μL per well, and then place the culture plates in a 37°C, 5% CO2 incubator for culture.

[0098] 8) After 6 hours, add 50 μL of selection medium to each well, and replace half of the medium with selection medium after 3 days.

[0099] 9) Observe the growth of hybridoma cells regularly, and when they grow to more than 1 / 10 of the bottom area of ​​the well, aspirate the supernatant for antibody detection.

[0100] 4. Hybridoma detection and cloning

[0101] 4.1 Take the culture supernatant of hybridoma cells and detect the antibody titer according to the titer detection method in the effect of water-soluble rapid immune adjuvant on the immunization of OVA-immunized mouse cells.

[0102] 4.2 Prepare mouse peritoneal macrophages as feeder cells.

[0103] 4.3 Prepare the hybridoma cell suspension to be cloned and dilute it to three different dilution degrees of 5, 10, and 20 cells per milliliter with HT medium containing 20% serum.

[0104] 4.4 Add peritoneal macrophages to the above-mentioned hybridoma cell suspension according to the proportion of 5×10 4 -1×10 5 cells per milliliter.

[0105] 4.5 Aliquot each type of hybridoma cells into one 96-well plate, with a volume of 100 μL per well.

[0106] 4.6 Incubate at 37 °C and 5% CO2 for 6 days. When visible clones appear, the antibodies can be detected; observe under an inverted microscope, mark the wells with only single clone growth, and take the supernatant for antibody detection.

[0107] 4.7 Expand the culture of the cells in the antibody detection positive wells, freeze a part of them, culture the other part, purify the antibodies using a Protein A purification column, and perform Elisa detection on the purified antibodies to verify whether they bind to the antigen.

[0108] Experimental results: A total of 21 hybridoma cell lines were obtained through hybridoma fusion and Elisa screening. After Elisa verification, the prepared monoclonal antibodies can all bind to the antigen GAPDH protein.

[0109] IV. Preparation of GAPDH rabbit polyclonal antibody using water-soluble rapid immune adjuvant

[0110] Use the water-soluble rapid immune adjuvant of Example 1 of the present invention to prepare GAPDH rabbit polyclonal antibody. The preparation method includes the following steps:

[0111] 1. Immunization of experimental rabbits

[0112] 1.1 Select two healthy New Zealand white rabbits at 6 weeks old (about 2 kg) and let them adapt to the new living environment for 1 week.

[0113] 1.2 Inject 0.5 mL of the antigen GAPDH into the two rabbits. It is more suitable to initially immunize 100 μg of antigen per rabbit, and 50 μg is sufficient for booster immunization. When the volume is insufficient, it can be supplemented with PBS.

[0114] 1.3 Mix 0.5 mL of the water-soluble rapid immune adjuvant with the prepared 0.5 mL of antigen, gently pipette to mix well, without emulsification.

[0115] 1.4 Carefully take out the rabbits from the cage. Immunize each rabbit at 4 sites (both the back of the neck and the root of the thigh are acceptable), and subcutaneously inject 250 μL at each site. After injection, pause for a few seconds to prevent antigen outflow.

[0116] 1.5 Boost immunization is carried out 3 weeks after the first immunization. Blood is collected 7 - 10 days after immunization (including blood collection for mid-course testing and final bleeding).

[0117] 2. Elisa titer detection

[0118] 2.1 Antigen coating: Pipette 100 μL of 1 μg / mL antigen GAPDH into each well of a 96-well plate, and incubate overnight at 4°C, or incubate at 37°C for 2 h.

[0119] 2.2 Blocking: Pour out the antigen solution, add 200 μL of blocking solution 2% BSA to each well, and incubate overnight at 4°C or incubate at 37°C for 2 h.

[0120] 2.3 Plate washing: Pour out the blocking solution, tap on the absorbent paper to drain the residual liquid as much as possible, wash the plate three times with the washing solution, and tap dry the residual liquid each time. If it needs to be stored for a while, dry it at 37°C and seal it with a sealed bag and store it at -20°C.

[0121] 2.4 Primary antibody incubation: Take the coated 96-well plate, add 100 μL of negative serum reference solution to the first well, add 1:2000 of the antiserum to be tested to the second well, and serially dilute the subsequent wells based on this. Incubate at 37°C for 1 h.

[0122] 2.5 Secondary antibody incubation: Pour out the liquid, wash the plate three times with the washing solution, and pat dry. Add 100 μL of HRP-labeled goat anti-rabbit IgG (diluted 1:5000) to each well, and incubate at 37°C for 45 min.

[0123] 2.6 TMB color development: Pour out the liquid, wash the plate three times with the washing solution, and pat dry. Add 100 μL of TMB substrate (self-made single-component TMB) to each well, and incubate at 37°C for 5 - 20 min (determine the color development time according to the color depth).

[0124] 2.7 Data analysis: Add 50 μL of stop solution (H2SO4) to each well, and read the absorbance value at a wavelength of 450 nm on an enzyme-linked immunosorbent assay reader.

[0125] 3. Preparation of affinity column

[0126] 3.1 Weigh 1 mg of CNBr-Sepherose 4B agarose gel and add it to 2 mmol / L hydrochloric acid. Incubate overnight at 4 °C to allow it to swell fully, and 3 mL of swollen gel can be obtained.

[0127] 3.2 Transfer the gel into a purification column and wash the medium 3 times with approximately 20 mL of 2 mmol / L hydrochloric acid.

[0128] 3.3 Wash the medium once with coupling buffer.

[0129] 3.4 Add 5 - 10 mg of antigen GAPDH dissolved in 5 mL of coupling buffer to the gel.

[0130] 3.5 Gently mix and shake at room temperature for 2 - 4 h, or overnight at 4 °C. If the binding efficiency needs to be determined, take a small amount of the solution for testing after this step, and wash the medium once with 20 mL of coupling buffer.

[0131] 3.6 Add 15 mL of 1% BSA solution and incubate at room temperature for 2 h or overnight at 4 °C.

[0132] 3.7 Wash the medium with phosphate buffer more than 3 times, with each wash being more than 15 mL.

[0133] 3.8 The antigen solid-phase immobilization is completed and can be used for purification.

[0134] 3.9 If it is not to be used immediately or after use, seal it with 20% ethanol.

[0135] 4. Purification and preservation of antiserum

[0136] 4.1 Dilute the antiserum with PBS in equal volume, centrifuge at 5000 - 10000 r / min for 15 min, and take the supernatant.

[0137] 4.2 Wash the antigen affinity column with 10 times the volume of PBS to balance the column.

[0138] 4.3 Add 10 mL of the diluted antiserum to the balanced column.

[0139] 4.4 Gently mix and shake at room temperature for 2 - 4 h, or overnight at 4 °C.

[0140] 4.5 Wash the antigen column with 10 times the volume of PBS to wash away the miscellaneous proteins bound to the column.

[0141] 4.6 Wash the column with 2 times the volume of antibody elution buffer (pH 2.5, 50 mmol / L glycine) to obtain specific antibodies.

[0142] 4.7 Balance the column with 10 times the volume of PBS.

[0143] 4.8 Seal the column with 20% alcohol and store at 4°C.

[0144] After obtaining the eluted antibody, concentrate it with sucrose or polyethylene glycol and then dialyze to remove salts in PBS. Measure the OD value of the antibody at a wavelength of 280 nm using a UV-visible spectrophotometer. Divide the obtained OD value by 1.35 to get the concentration of the measured antibody. Add 40 - 50% glycerol and store at -20°C for long-term preservation.

[0145] Experimental results: After detecting the titer of antiserum during the immunization process, it was found that the titer of antiserum in immunized rabbits was above 100,000 on the 35th day. The purified polyclonal antibody showed excellent detection effects in WB, IHC, and IF applications.

[0146] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, 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. A water-soluble rapid immune adjuvant, characterized in that: Taking the preparation of 1 L of adjuvant as an example, the water-soluble rapid immune adjuvant comprises 50 - 500 μg / mL of 7α,25-OHC, 1% - 3% of cyclodextrin, 1 - 5 mg / mL of aluminum salt, 5% - 7% of chitosan-based thermosensitive hydrogel, and the balance being deionized water; The aluminum salt is aluminum hydroxide; The chitosan-based thermosensitive hydrogel is conjugated with imidazoquinoline; The imidazoquinoline is R848; The preparation method for conjugating the chitosan-based thermosensitive hydrogel with R848 comprises the following steps: S1, adding chitosan powder into deionized water, ultrasonically dispersing uniformly, then dropwise adding lactic acid while stirring until the chitosan is completely dissolved, adding bromoacetic acid to adjust the pH to 7 - 7.5, and reacting at 20 - 30 °C for 2 h to obtain carboxylated chitosan; S2, sequentially adding EDC, NHS, and R848 into the carboxylated chitosan, and reacting at 20 - 30 °C for 1 - 1.5 h; then placing the reaction solution in an environment at 4 °C, adding an aqueous solution of β-glycerophosphate with a mass concentration of 10.8%, and mixing uniformly to obtain the chitosan-based thermosensitive hydrogel conjugated with R848.

2. The water-soluble rapid immune adjuvant according to claim 1, characterized in that: The deacetylation degree of the chitosan powder is 75% - 99%, the molecular weight is 10 kd - 500 kd, the viscosity is 30 - 100 cP, and the mass ratio of the chitosan to deionized water is (1 - 2):

100.

3. The water-soluble rapid immune adjuvant according to claim 1, characterized in that: The molar ratio of the chitosan to bromoacetic acid in step S1 is (1 - 3):

20.

4. The water-soluble rapid immune adjuvant according to claim 1, characterized in that: The molar ratio of the carboxylated chitosan:EDC:NHS:R848 in step S2 is 500:(1 - 2):(1 - 3):

25.

5. A preparation method of the water-soluble rapid immune adjuvant according to any one of claims 1-4, characterized in that: It includes the following steps: Dissolve 7α,25-OHC, cyclodextrin, aluminum salt and chitosan-based thermosensitive hydrogel in deionized water, and sterilize it with steam at 1.23×10 5 Pa for 30 min to obtain it.

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