Preparation method of high-titer immunocompetence SlgA polyclonal antibody

Abstract

The invention relates to the technical field of biology, in particular to a preparation method of a high-titer immunocompetence SlgA polyclonal antibody. The invention discloses a preparation method of a high-titer immunocompetence SlgA polyclonal antibody, which comprises the following steps: (1) emulsifying a bovine colostrum SIgA antigen with the purity of more than 95% to obtain an antigen emulsion; (2) injecting an immune animal with the antigen emulsion for multiple times to obtain SIgA antiserum; (3) purifying the SIgA antiserum to obtain an SIgA polyclonal antibody; the SIgA antigen is extracted from bovine coloctrum, the high-titer rabbit anti-bovine SIgA polyclonal antibody is successfully prepared through the method, the antigen immunization dosage is reduced, meanwhile, the high-titer antibody can be obtained, the immune double diffusion titer of the antibody is 1: 16, the ELISA titer is larger than 512000, and the method is a new effective antibody preparation way.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a method for preparing a high-titer immunomodulatory SlgA polyclonal antibody. Background Technology

[0002] SIgA is an important antibody found in exocrine fluids and a major effector factor in mucosal immunity. In immune responses in the respiratory and digestive tracts, it not only provides an immune barrier to the mucosal surface, preventing microbial adhesion and neutralizing toxins, but also binds to antigens in the lamina propria of the mucosa, preventing the formation of local immune complexes. Furthermore, it has anti-inflammatory, regulatory, and opsonizing effects, playing a crucial role in mucosal immunity through both active and passive immunization. Simultaneously, it protects the gastrointestinal mucosa and resists the action of proteolytic enzymes, preventing its inactivation by digestion.

[0003] Currently, methods for detecting SIgA in bovine colostrum include rocket electrophoresis, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunogold assay. These methods all fall under immunological detection and rely heavily on the key technology of specific antibody preparation. However, to date, no commercial sales information for bovine colostrum SIgA standards and related antibodies has been found, limiting the development and utilization of bovine colostrum SIgA products. While there are reports of bovine colostrum SIgA isolation and purification in China, the SIgA purity is only 85.3%, which does not meet the purity requirements for antigens in immunized animals. HPLC molecular sieving methods have also been used in SIgA detection research, but the SIgA standards used are derived from human colostrum and cannot be used for immunological detection. The large-scale preparation of high-purity SIgA has become a technical bottleneck restricting immunological detection methods. Previously, our company, Jiangsu Tianmeijian Natural Biotechnology Co., Ltd., applied for a patent related to the preparation of high-purity bovine colostrum SIgA technology, application number CN202410104203.9. We prepared bovine colostrum SIgA with a purity of ≥95% at the milligram level. Based on this, we prepared high-titer SIgA antiserum for animals, which can provide theoretical and experimental basis for the establishment of immunological detection methods for bovine colostrum SIgA and the research and development of related products.

[0004] Among existing antiserum preparation techniques, the preparation techniques for animal-derived IgG polyclonal antibodies and monoclonal antibodies are relatively mature. The preparation of milk-derived SIgA polyclonal antibodies has only been reported with porcine colostrum; to date, no reports have been found regarding the preparation of high-titer SIgA polyclonal antibodies from bovine colostrum. In animal immunization, the choice of immunization dose is crucial. Generally, obtaining high-titer antibodies requires a certain immunization dose, which inevitably increases the demand for the corresponding antigen. This is especially problematic for antigens that are difficult to prepare. Therefore, reducing the antigen immunization dose while still obtaining high-titer antibodies represents an effective new approach for antibody preparation. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this application provides a method for preparing a high-titer immunogenic SlgA polyclonal antibody.

[0006] This application discloses a method for preparing a high-titer immunomodulatory polyclonal antibody against SIgA, successfully producing a high-titer rabbit anti-bovine SIgA polyclonal antibody with an immunodiffusion titer of 1:16 and an ELISA titer greater than 512,000. Compared with existing antiserum preparation techniques, this application uses a lower dose of 0.2–0.4 mg / animal for animal immunization and appropriately extends the immunization time. After six immunizations, it was found that not only was immune paralysis not produced, but a high-titer antibody was also obtained, saving valuable antigen usage. The antiserum obtained after animal immunization was preliminarily purified and then freeze-dried into antibody lyophilized powder. The prepared lyophilized antibody powder has a stable titer and completely retains the activity of the SIgA antibody, providing an effective method for long-term antibody preservation.

[0007] In a first aspect, this application provides a method for preparing a high-titer immunologically active SlgA polyclonal antibody, employing the following technical solution: A method for preparing a high-titer immunomodulatory polyclonal antibody against SlgA includes the following steps: (1) Emulsify bovine colostrum SIgA antigen with a purity greater than 95% to obtain an antigen emulsion; (2) The antigen emulsion was injected into the immunized animals multiple times to obtain SIgA antiserum; (3) The SIgA antiserum was purified to obtain SIgA polyclonal antibody.

[0008] By adopting the above technical solution, bovine colostrum SIgA antigen with a purity greater than 95% was extracted from bovine colostrum in this application, and a high-titer rabbit anti-bovine SIgA polyclonal antibody was successfully prepared by the method of this application. The antibody's immunodiffusion titer was 1:16, and the ELISA titer was greater than 512000.

[0009] Preferably, the specific steps of step (2) are as follows: The antigen emulsion is injected into the immunized animal for initial immunization. Multiple booster immunizations were administered after the initial immunization. Blood was collected 7–10 days after the last immunization, serum was separated, and potency was tested.

[0010] Preferably, the initial immunization dose is 0.2-0.4 ml per animal, with a concentration of 400 μg / 0.5 mL; the booster immunization dose is 0.2-0.4 ml per animal, with a concentration of 200 μg / 0.5 mL; and the final immunization dose is 0.2-0.4 ml per animal, with a concentration of 400 μg / 0.5 mL.

[0011] By adopting the above technical solution, the selection of the animal immunization dose is crucial in existing antiserum preparation technologies. Excessively high or low doses can affect the body's immune response. Repeated low-dose injections can cause immune paralysis and inhibit antibody production. To avoid immune paralysis, the immunization dose for animals is generally chosen to be 1–2 mg / animal. This application uses a lower dose of 0.2–0.4 mg / animal and appropriately prolongs the immunization time. After six immunizations, it was found that not only was immune paralysis not produced, but high-titer antibodies were also generated. The method of this application not only avoids the occurrence of immune paralysis but also saves valuable antigen usage and obtains high-titer antibodies.

[0012] This application employs multiple immunization responses, reduces the immunization dose, prolongs the immunization time, reduces animal immune rejection, and makes the animal immunization dose lower than the conventional dose, thus reducing the amount of standard material used; it also has a higher potency, reaching 4 times the potency of commercially available immunization standards.

[0013] The preferred method for preparing the antigen emulsion includes the following steps: For the initial immunization, the SIgA antigen was emulsified using Freund's complete adjuvant to obtain an antigen emulsion; During booster and final immunizations, SIgA antigens are emulsified using Freund's incomplete adjuvant to obtain antigen emulsions.

[0014] Preferably, the volume ratio of SIgA antigen to Freund's complete adjuvant is 1:(3-6), and the volume ratio of SIgA antigen to Freund's incomplete adjuvant is 1:(3-6).

[0015] Preferably, the injections are administered in 6 doses, with 5 booster immunizations given after the initial immunization, and immunizations given 7 days apart; the details are as follows: First injection (initial immunization): Inject the immunized animals with the above-mentioned antigen emulsion; the immunization sites are multiple subcutaneous injections on the limbs and back of the rabbits; Second injection (boost immunization): The second injection is given 7 days after the first injection. The immunization sites are the subcutaneous tissue of the neck and back and the axillary lymph nodes. Third injection (boost immunization): The third injection is given 7 days after the second injection. The immunization sites are the subcutaneous lymph nodes in the back of the neck and the axillary lymph nodes. Fourth injection (boost immunization): The fourth injection is given 7 days after the third injection. The immunization sites are the subcutaneous tissue of the neck and back and the axillary lymph nodes. Fifth injection (boost immunization): Seven days after the fourth injection, the fifth injection will be given, and the immunization sites will be the subcutaneous tissue of the neck and back and the axillary lymph nodes. The sixth injection (final immunization): The sixth injection is given 7 days after the fifth injection, and the immunization sites are multiple subcutaneous injections on the back of the neck.

[0016] Serum titer test: Blood was collected 7–10 days after the sixth injection, serum was separated, and titer was tested.

[0017] Preferably, the immunized animal is a New Zealand white rabbit.

[0018] Preferably, the antibody purification step in step (3) is as follows: add saturated ammonium sulfate solution to SIgA antiserum to make SIgA antiserum reach 40% saturation, centrifuge, add saturated ammonium sulfate solution again to make SIgA antiserum reach 35% saturation, centrifuge, and obtain SIgA polyclonal antibody.

[0019] In one specific feasible implementation, the antibody purification steps are as follows: Slowly add saturated ammonium sulfate solution to SIgA antiserum to achieve 40% saturation, stirring continuously to prevent excessive local concentration. Then, incubate overnight at 4°C. Centrifuge at 4000 rpm for 15 min, collect the precipitate, dissolve the precipitate in PBS with half the volume of SIgA antiserum (i.e., half the initial volume of added SIgA antiserum), add saturated ammonium sulfate solution to achieve 35% saturation, centrifuge at 4000 rpm for 15 min, collect the precipitate, dissolve the precipitate in PBS with an equal volume of serum (i.e., the same volume as the initial added SIgA antiserum), and dialyze with the same buffer PBS until no ammonium ions are detected. Centrifuge at 10000 rpm for 15 min; the supernatant is the purified SIgA antiserum, which is the SIgA polyclonal antibody.

[0020] Preferred method for preserving SIgA polyclonal antibodies, wherein the method is any one of the following: Method 1: Freeze-dry the SIgA polyclonal antibody to prepare a lyophilized powder, and store it in a sealed container at -20°C; Preferably, the SIgA polyclonal antibody is freeze-dried using the following steps: the SIgA polyclonal antibody is frozen at -60 to -40°C for 2 to 4 hours, then the vacuum is turned on, and the temperature is adjusted to -45 to -35°C for 0.5 to 1 hour. After that, the temperature is adjusted to -10 to -5°C for 6 to 12 hours, and then the temperature is adjusted to 20 to 25°C for 3 to 5 hours to obtain the freeze-dried powder.

[0021] In a specific feasible implementation, the antibody lyophilization steps are as follows: SIgA polyclonal antibody is lyophilized on a YWLG-50F lyophilizer. The lyophilization parameters are set as follows: In the pre-freezing stage, the SIgA polyclonal antibody is frozen at -40°C for 2–4 hours. The vacuum and heating control system is turned on, and the antibody is held at -35°C for 0.5–1 hour before entering the sublimation drying stage. The temperature is controlled at -10–-5°C for 6–12 hours. In the desorption drying stage, the temperature is controlled at 20–25°C for 3–5 hours until the sample temperature and shelf temperature are consistent. The lyophilization process is then completed, yielding a high-titer SIgA polyclonal antibody lyophilized powder. The lyophilized powder is a milky white, glossy crystalline powder. The titer before and after lyophilization is consistent, as determined by immunodiffusion.

[0022] Method 2: After liquid SIgA polyclonal antibody is aliquoted, preservatives are added and stored at -20°C. The titer remains stable within one year, but repeated freeze-thaw cycles should be avoided to prevent the titer from decreasing.

[0023] By adopting the above technical solution, this application pre-purifies the antiserum, removes a large number of non-antibody components, and then uses freeze-drying technology to prepare antibody lyophilized powder, and explores the freeze-drying preparation process conditions of the antiserum; the antiserum lyophilized powder prepared in this application has stable titer and completely retains the activity of SIgA antibody, providing an effective method for long-term antibody preservation.

[0024] In summary, this application includes at least one of the following beneficial technical effects: 1. This application discloses a method for preparing a high-titer immunomodulatory SlgA polyclonal antibody, which successfully prepared a high-titer SlgA polyclonal antibody with an immunodiffusion titer of 1:16 and an ELISA titer greater than 512000; 2. This application uses a low dose of 0.2-0.4 mg / animal for immunization and appropriately prolongs the immunization time. After 6 immunizations, it was found that not only was immune paralysis not produced, but high-titer antibodies were also produced. The method of this application not only avoids the occurrence of immune paralysis, but also saves valuable antigen usage and obtains high-titer antibodies. 3. In this application, the antiserum was initially purified to remove a large number of non-antibody components, and then prepared into antibody lyophilized powder using freeze-drying technology. The freeze-drying preparation process conditions of the antiserum were also explored. The prepared antiserum lyophilized powder has a stable titer and completely retains the activity of SIgA antibody, providing an effective method for long-term antibody preservation. Detailed Implementation

[0025] The technical solutions of this application are further illustrated by specific embodiments below. These specific embodiments do not represent a limitation on the scope of protection of this application. Any non-essential modifications and adjustments made by others based on the concept of this application still fall within the scope of protection of this application.

[0026] All raw materials involved in this application are commercially available products, among which, Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma. The coating diluent was purchased from Solarbio. The primary antibody (goat anti-bovine) and secondary antibody (goat anti-rabbit IgG, HRP) were purchased from Solarbio. In the embodiments of this application, the pH of the saturated ammonium sulfate solution was adjusted to 7.0, and the PBS concentration was 10 mmol / L and the pH was 7.4 unless otherwise specified.

[0027] The present application will be further described in detail below with reference to the embodiments.

[0028] Example 1: Obtaining SIgA antigen

[0029] A method for rapidly preparing high-purity secretory immunoglobulin A from bovine colostrum, comprising the following steps: (1) Centrifugation and defatting: Take 1 kg of bovine colostrum on the 3rd day (i.e. the 3rd day after the cow produces milk), centrifuge at 4000 r / min for 30 min at 4℃ to remove the cell precipitate at the bottom and the floating fat layer. Filter the remaining part through 8 layers of gauze to obtain colostrum whey for later use. (2.1) First ammonium sulfate precipitation: After diluting the colostrum whey obtained in step (1) with physiological saline, add saturated ammonium sulfate solution to the diluted solution and stir continuously until the ammonium sulfate reaches 50% saturation. Then, refrigerate and let stand overnight. After centrifugation at 6000 r / min for 10 min, discard the supernatant and keep the ammonium sulfate precipitate for later use. In this step, the volume ratio of physiological saline used for dilution to colostrum whey is 1:1. (2.2) Second ammonium sulfate precipitation: Dissolve the ammonium sulfate precipitate from step (2.1) with PBS, then add saturated ammonium sulfate solution to make the ammonium sulfate saturation reach 40%, then refrigerate at 4°C for 4 hours, and centrifuge at 5000 r / min for 12 min to obtain the supernatant; in this step, the PBS used to dissolve the precipitate has the same volume as the physiological saline used for dilution in step (2.1).

[0030] (2.3) Third ammonium sulfate precipitation: Add saturated ammonium sulfate solution to the supernatant obtained in step (2.2) to make the ammonium sulfate saturation reach 35%. After centrifugation at 4000 r / min for 15 min, discard the supernatant and obtain the precipitate for later use. (3) Gel column desalting: The precipitate obtained in step (2.3) was dissolved in PBS containing 0.15 mol / L sodium chloride (the original concentration of PBS was 10 mmol / L, pH 7.4), and then centrifuged at 10000 r / min for 10 min. The supernatant was added to a Sephadex G-25 column (5×20 cm) for desalting, eluted with PBS without sodium chloride, and the eluted protein peak was collected. (4) Ultrafiltration concentration: The protein peaks collected in step (3) are concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 100kDa. During the ultrafiltration concentration process, PBS with a volume equivalent to 10 times the total volume of the protein peaks is added for replenishment. The ultrafiltration concentration is completed when the volume is 30mL, and the ultrafiltration concentrate is obtained. (5) Sephacryl S-300 column chromatography: Add saturated ammonium sulfate solution to the ultrafiltration concentrate in step (4) to make the ammonium sulfate saturation reach 35%. After refrigerating and standing for 4 hours, centrifuge at 10000 r / min for 10 min. Dissolve the precipitate with 5 mL of PBS. After centrifuging again at 10000 r / min for 10 min, add the supernatant to the Sephacryl S-300 column (2.6 × 90 cm) and elute with PBS. Collect 5 mL per tube. Control the flow rate at 0.5 mL / min. Collect the second protein peak that flows out. Combine the protein peaks and dialyze to the binding buffer (20 mmol / L PBS, pH 7.0, containing 0.15 mol / L NaCl) to obtain the dialysate. (6) Protein G column separation: Centrifuge the dialysate from step (5) at a rate of 5000 r / min for 10 min, add it to the Protein G column (5 mL) at a rate of 1 mL / min, collect the breakthrough peak, combine the breakthrough peaks, add saturated ammonium sulfate solution to 35% saturation to obtain a mixture, and refrigerate the mixture overnight. (7) Superdex 200 column chromatography separation: The mixture after standing overnight was centrifuged at 10000 r / min for 10 min. The ammonium sulfate precipitate was dissolved in PBS (PBS to mixture volume ratio 1:1). It was added to a Superdex 200 column (1.6×90cm) for chromatography separation. Elution was performed at 1 mL / min. The second elution peak was collected. Then, it was subjected to ultrafiltration desalting concentration and lyophilization to obtain secretory immunoglobulin A with a purity greater than 95% and a molecular weight of 496.42±3.42kDa. The relative yield was 125.3%.

[0031] Example 2: Antibody Preparation 1. Animal selection: Select healthy New Zealand white rabbits weighing 2.2-2.5 kg. Vaccinate the animals, and during the immunization process, ensure that the animals' nutritional status and the hygiene of their living environment are maintained in good condition and meet the requirements of animal husbandry and management regulations.

[0032] 2. Antigen emulsification and dosage: 2.1 Antigen emulsification: Antigen emulsion used for initial immunization: Bovine colostrum SIgA antigen (prepared in Example 1) was dissolved in PBS at a concentration of 400 μg / 0.5 mL. Freund's complete adjuvant (the volume ratio of SIgA antigen to Freund's complete adjuvant was 1:4) was taken and connected through the antigen emulsification linker. The antigen and adjuvant were quickly mixed to achieve complete emulsification.

[0033] Antigen emulsions used for booster immunization: Bovine colostrum SIgA antigen (prepared in Example 1) was dissolved in PBS at a concentration of 200 μg / 0.5 mL. Freund's incomplete adjuvant (the volume ratio of SIgA antigen to Freund's incomplete adjuvant was 1:4) was taken and connected through the antigen emulsification linker to quickly mix the antigen and adjuvant to make them completely emulsified.

[0034] Antigen emulsion used in the last immunization: Bovine colostrum SIgA antigen (prepared in Example 1) was dissolved in PBS at a concentration of 400 μg / 0.5 mL. Freund's incomplete adjuvant (the volume ratio of SIgA antigen to Freund's incomplete adjuvant was 1:4) was taken and connected through the antigen emulsification linker to quickly mix the antigen and adjuvant to achieve complete emulsification.

[0035] 2.2 Dosage of Emulsified Antigen For the initial immunization, an antigen emulsion (prepared using Freund's complete adjuvant) was used, with an immunization dose of 0.3 ml per animal; For booster and final immunization, antigen emulsion (prepared with Freund's incomplete adjuvant) was used. The booster dose was 0.3 ml per animal, and the final immunization dose was 0.3 ml per animal.

[0036] 3. Animal immunization: Five booster immunizations were administered after the initial immunization, with a 7-day interval between each booster immunization, for a total of six immunizations.

[0037] The initial immunization was performed by subcutaneous injection of antigen emulsion (prepared with Freund's complete adjuvant) at multiple sites on the limbs and back of the rabbits. One week later, a booster immunization was performed using antigen emulsion (prepared with Freund's incomplete adjuvant) at subcutaneous sites on the back of the neck and axillary lymph nodes. Thereafter, booster immunizations were performed every 7 days at the same dose and via the same route. The final immunization was performed by subcutaneous injection of antigen emulsion (prepared with Freund's incomplete adjuvant) at multiple sites on the back of the neck.

[0038] 4. Blood collection and antiserum separation: Seven days after the last immunization, blood was collected from the marginal ear vein. Antibody titers were detected using the double diffusion method and ELISA. Once the titers met the requirements, blood was collected from the carotid artery on an empty stomach. The blood volume was maintained at 1% of the animal's body weight over the past two weeks (i.e., 10% of the total blood volume). The blood was then transferred to a 4°C refrigerator and left overnight to allow for complete serum separation. The following day, the serum was centrifuged at 3500 rpm for 15 minutes, and the supernatant was collected as SIgA antiserum.

[0039] 5. Antibody purification: Slowly add saturated ammonium sulfate solution to SIgA antiserum to achieve 40% saturation, stirring constantly to prevent excessive local concentration. Then, incubate overnight at 4°C. Centrifuge at 4000 rpm for 15 min, collect the precipitate, and dissolve it in PBS with half the volume of SIgA antiserum (i.e., half the volume of the SIgA antiserum prepared in step 4). Slowly add saturated ammonium sulfate solution to achieve 35% saturation, stirring constantly to prevent excessive local concentration. Centrifuge at 4000 rpm for 15 min, collect the precipitate, and dissolve it in PBS with an equal volume of SIgA antiserum (i.e., an equal volume of the SIgA antiserum prepared in step 4). Dialyze with PBS buffer until no ammonium ions are detected. Centrifuge at 10000 rpm for 15 min; the supernatant is the purified SIgA antiserum, which is the SIgA polyclonal antibody.

[0040] Example 3: Determination of antibody titer by double diffusion immunoassay 1) Take 3 ml of melted 1.2% agarose solution (prepared at 60℃) and quickly pour it onto a glass slide that has been leveled. After solidification, use a 3-5 mm perforating tube to punch holes in a quincunx pattern. Generally, there is one hole in the center and six holes around the perimeter, with a hole spacing of 5 mm.

[0041] 2) Add 10 µl (1 mg / ml) of SlgA antigen solution (prepared in Example 1) to the center well, and add 10 µl of SlgA antiserum (prepared in step 5 of Example 2) diluted in equal multiples (1:2, 1:4, 1:8, 1:16, 1:32, 1:64) to the surrounding 6 wells in sequence. Then place the agar plate in a humidified chamber and let it stand at room temperature for 38 h. Observe the white precipitation line that appears to determine the titer of the antiserum.

[0042] 3) A precipitation line will appear between the central well and the corresponding diluted antiserum well. The lowest dilution factor of the antiserum is the antiserum titer. When the titer reaches 1:16 or higher, cardiac blood can be collected to collect antiserum. If the precipitation line is unclear, it can be stained with Coomassie Brilliant Blue R-250 staining solution for 10 minutes, then destained with destaining solution before observation.

[0043] 4) The titer of SIgA polyclonal antibody was determined to be 1:16 by the double diffusion immunoassay.

[0044] Example 4: ELISA method for determining antibody titer

[0045] 1) Coating: Dilute SIgA antiserum (prepared in step 5 of Example 2) to the required concentration with coating diluent, mix well and add 100 μL to each well of the microplate. Cover with the cover film and incubate overnight at 4°C. The next day, remove the microplate, wash it 3 times, and pat dry for later use.

[0046] 2) Blocking: Add 200 μL of blocking buffer to each well of the microplate and incubate at 37°C for 2 hours. Remove the microplate, wash it once, and pat it dry before use.

[0047] 3) Add primary antibody: Add the corresponding primary antibody (goat anti-bovine) to the coated ELISA plate and incubate at 37°C for 1 hour; remove the ELISA plate, wash it 3 times, and pat it dry.

[0048] 4) Add secondary antibody: Add enzyme-labeled secondary antibody (goat anti-rabbit IgG, HRP) to the microplate, 100 uL per well, incubate at 37℃ for 1 h, remove the microplate, wash the plate and pat dry.

[0049] 5) Color development and termination: Add 100 μL of color development solution to the microplate, incubate at room temperature for 15 min, add 50 μL of 1M HCl to terminate the reaction, and read the value at 450 nm on the microplate reader in time. The results are shown in Table 1.

[0050] Table 1. Titer of SIgA polyclonal antibody determined by ELISA

[0051] The data in Table 1 show that the titer of SIgA polyclonal antibody determined by ELISA is greater than 1:512000, indicating that the SIgA polyclonal antibody prepared according to the method of this application has a very high titer.

[0052] This application discloses a method for preparing a high-titer, immunologically active SIgA polyclonal antibody. The method successfully prepared a high-titer SIgA polyclonal antibody with an immunodiffusion titer of 1:16 and an ELISA titer greater than 512,000. Using a low dose of 0.2–0.4 mg / animal and appropriately extending the immunization time, after six immunizations, it was found that not only was immune paralysis not produced, but high-titer antibodies were also generated. The method of this application not only avoids the occurrence of immune paralysis but also saves valuable antigen usage, achieving a high-titer antibody.

[0053] Example 5: Antibody Lyophilization

[0054] The SIgA polyclonal antibody prepared in step 5 of Example 2 was lyophilized, and the specific steps are as follows: SIgA polyclonal antibody was freeze-dried in a YWLG-50F freeze dryer. The freeze-drying parameters were set as follows: In the pre-freezing stage, SIgA polyclonal antibody was pre-frozen at -40℃ for 3 hours, and the vacuum and heating control system was turned on. After maintaining the temperature at -35℃ for 0.8 hours, the sublimation drying stage was started, and the temperature was controlled at -7℃ for 10 hours. In the desorption drying stage, the temperature was controlled at 23℃ for 4 hours until the sample temperature and the shelf temperature were consistent. The freeze-drying was then stopped, and high-titer SIgA polyclonal antibody freeze-dried powder was obtained. The freeze-dried powder was milky white and glossy crystals.

[0055] The titers before and after lyophilization were consistent, as determined by the immunodiffusion method.

[0056] The titer of the SIgA polyclonal antibody after lyophilization was determined by the double diffusion immunoassay to be 1:16, which was consistent with the titer after lyophilization.

[0057] Example 6: Antibody Preservation 1. Freeze-drying preservation

[0058] The freeze-dried powder prepared in Example 5 was sealed and stored at -20°C, and the potency of the freeze-dried powder was tested. The results are shown in Table 2.

[0059] Table 2. Potency of lyophilized powders stored in sealed containers at -20℃

[0060] As shown in Table 2, preparing SIgA polyclonal antibodies into lyophilized powder and storing them can ensure that the titer of SIgA polyclonal antibodies remains stable within 5 years.

[0061] 2. Preservatives should be added. After the liquid SIgA polyclonal antibody prepared in step 5 of Example 2 was aliquoted, a preservative (sodium azide) was added and mixed, and the mixture was stored at -20°C. The titer of the SIgA polyclonal antibody was then tested, and the results are shown in Table 3. The preservative accounted for 0.03% of the total mass of the system.

[0062] Table 3. Titer of liquid SIgA polyclonal antibody stored at -20℃

[0063] As shown in Table 3, the titer of liquid SIgA polyclonal antibody with added preservatives remains stable within one year. During storage, repeated freeze-thaw cycles should be avoided to prevent the titer from decreasing.

[0064] This application involves the preliminary purification of antiserum to remove a large number of non-antibody components, followed by freeze-drying to prepare lyophilized antibody powder. The freeze-drying process conditions for the antiserum were also explored. The prepared lyophilized antiserum powder has a stable titer and fully retains the activity of SIgA antibody, providing an effective method for the long-term preservation of antibodies.

Claims

1. A method for preparing a high-titer immunomodulatory polyclonal antibody against SlgA, characterized in that: Includes the following steps: (1) Emulsify bovine colostrum SIgA antigen with a purity greater than 95% to obtain an antigen emulsion; (2) The antigen emulsion was injected into the immunized animals multiple times to obtain SIgA antiserum; (3) The SIgA antiserum was purified to obtain SIgA polyclonal antibody.

2. The method for preparing a high-titer immunomodulatory SlgA polyclonal antibody according to claim 1, characterized in that: The specific steps of step (2) are as follows: The antigen emulsion is injected into the immunized animal for initial immunization. Multiple booster immunizations were administered after the initial immunization. Blood was collected 7–10 days after the last immunization, serum was separated, and potency was tested.

3. The method for preparing a high-titer immunomodulatory SlgA polyclonal antibody according to claim 2, characterized in that: The initial immunization dose is 0.2–0.4 ml per animal, with a concentration of 400 μg / 0.5 mL; the booster immunization dose is 0.2–0.4 ml per animal, with a concentration of 200 μg / 0.5 mL; and the final immunization dose is 0.2–0.4 ml per animal, with a concentration of 400 μg / 0.5 mL.

4. The method for preparing a high-titer immunomodulatory SlgA polyclonal antibody according to claim 2, characterized in that: The preparation method of the antigen emulsion includes the following steps: For the initial immunization, the SIgA antigen was emulsified using Freund's complete adjuvant to obtain an antigen emulsion; During booster and final immunizations, SIgA antigens are emulsified using Freund's incomplete adjuvant to obtain antigen emulsions.

5. The method for preparing a high-titer immunomodulatory SlgA polyclonal antibody according to claim 4, characterized in that: The volume ratio of the SIgA antigen to Freund's complete adjuvant is 1:(3-6), and the volume ratio of the SIgA antigen to Freund's incomplete adjuvant is 1:(3-6).

6. The method for preparing a high-titer immunomodulatory SlgA polyclonal antibody according to claim 2, characterized in that: Five booster immunizations are administered after the initial immunization, with each immunization given 7 days apart.

7. The method for preparing a high-titer immunomodulatory SlgA polyclonal antibody according to claim 2, characterized in that: During the initial immunization, the immunization sites are multiple subcutaneous injections on the rabbit's limbs and back; during the booster immunization, the immunization sites are subcutaneous injections on the back of the neck and axillary lymph nodes; during the final immunization, the immunization sites are multiple subcutaneous injections on the back of the neck.

8. The method for preparing a high-titer immunomodulatory SlgA polyclonal antibody according to claim 1, characterized in that: The immunized animal was a New Zealand white rabbit.

9. The method for preparing a high-titer immunologically active SlgA polyclonal antibody according to claim 1, characterized in that: The antibody purification steps in step (3) are as follows: add saturated ammonium sulfate solution to SIgA antiserum to make SIgA antiserum reach 40% saturation, centrifuge, add saturated ammonium sulfate solution again to make SIgA antiserum reach 35% saturation, centrifuge, and obtain SIgA polyclonal antibody.

10. The method for preparing a high-titer immunomodulatory SlgA polyclonal antibody according to claim 1, characterized in that: The SIgA polyclonal antibody shall be preserved using any of the following methods: Method 1: SIgA polyclonal antibody was freeze-dried to prepare lyophilized powder; The freeze-drying process is as follows: freeze the SIgA polyclonal antibody at -60 to -40°C for 2 to 4 hours, turn on the vacuum, adjust the temperature to -45 to -35°C and freeze for 0.5 to 1 hour, then adjust the temperature to -10 to -5°C and freeze for 6 to 12 hours, and finally adjust the temperature to 20 to 25°C and freeze for 3 to 5 hours to obtain the freeze-dried powder. Method 2: Add preservatives to the SIgA polyclonal antibody for preservation.

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