Preparation method of anti-human hepatitis B surface antigen monoclonal antibody

Abstract

The invention discloses a preparation method of an anti-human hepatitis B surface antigen monoclonal antibody. The preparation method comprises the following steps: step 1, extracting a human hepatitis B surface antigen HBsAg; 2, selecting an experimental animal, wherein the experimental animal is a mouse; 3, animal immunization is conducted on the mouse, purified antigen HBsAg is injected for immunization, and injection positions are subcutaneous and abdominal cavities; step 4, continuing to carry out second immunization and third immunization by using the same dose of antigen and the same method; step 5, using the same dosage of antigen and the same method to carry out booster immunization before cell fusion; 6, taking out the spleen of the mouse after three days of boosted immunization, preparing a cell suspension, preparing myeloma cells, and fusing the cell suspension and the myeloma cells into hybridoma cells; 7, screening hybridoma cells, and culturing and cloning the screened hybridoma cells; and 8, preparing ascites, and purifying the ascites to obtain the monoclonal antibody. Compared with the prior art, the invention has the advantage of providing the preparation method of the anti-human hepatitis B surface antigen monoclonal antibody with higher purity.

Description

Technical Field

[0001] This invention relates to cell engineering and antibody engineering technology, specifically to a method for preparing monoclonal antibodies against human hepatitis B surface antigen. Background Technology

[0002] Hepatitis B is a serious disease caused by hepatitis B virus (HBV) infection, posing a significant threat to human health. It is highly contagious, extremely harmful, and difficult to cure. More than half of the world's chronic HBV carriers are Chinese. Recent large-scale population surveys indicate that the HBsAg detection rate in my country is approximately 10%, and the prevalence of HBV, including anti-HBs and anti-HBc, is 50-60%. Some data estimate that there may be over 120 million chronic asymptomatic HBV carriers (AsC) in my country, with 28 million currently diagnosed with hepatitis B, a prevalence rate of approximately 2770 / 100,000, an annual incidence rate of 230 / 100,000, and a chronic hepatitis B prevalence rate of 0.1-1%. Cumulatively, both current and past cases, more than half of my country's population has been infected with HBV. While the widespread implementation of hepatitis B vaccination among newborns in my country has shown promise in controlling HBV, the current situation remains severe. Therefore, research into new drugs and methods for the prevention and treatment of hepatitis B is of particular importance in my country.

[0003] Hepatitis B surface antigen (HBsAg) is the outer shell protein of the hepatitis B virus. As an expression of the hepatitis B virus S gene, it belongs to the category of viral envelope proteins. Although it is not infectious itself, it is the first serum viral antigen to appear in later disease intervention. Its basic structure is closely related to the HBV template and it has a high correlation with DNA levels. Therefore, the value of HBsAg research is analyzed.

[0004] Passive antibody immunization can effectively prevent and treat viral infections. The preparation of monoclonal antibodies against human hepatitis B surface antigen mainly involves methods such as in vivo ascites in mice, in vitro culture of hybridoma cells, and genetic engineering. Among these, the in vivo ascites in mice method is widely used due to its low cost, high yield, and relatively simple production process, allowing for large-scale production in a short period; however, it contains more impurities. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the above-mentioned technical defects and provide a method for preparing a monoclonal antibody against human hepatitis B surface antigen with higher purity.

[0006] To solve the above-mentioned technical problems, the present invention provides a method for preparing monoclonal antibodies against human hepatitis B surface antigen, comprising the following steps: Step 1: Extracting human hepatitis B surface antigen HBsAg; Step 2: Selecting experimental animals, specifically mice; Step 3: Immunizing mice by injecting purified HBsAg antigen subcutaneously and intraperitoneally; Step 4: Performing a second and third immunization using the same dose of antigen and the same method; Step 5: Performing a booster immunization using the same dose of antigen and the same method before cell fusion; Step 6: Three days after the booster immunization, removing the spleen from the mice to prepare a cell suspension, preparing myeloma cells, and fusing the cell suspension with myeloma cells to form hybridoma cells; Step 7: Screening hybridoma cells and culturing clones of the screened hybridoma cells; Step 8: Preparing ascites fluid and purifying the ascites fluid to obtain monoclonal antibodies.

[0007] Preferably, the experimental animals in step 2 are 8-12 week old BALB / C mice.

[0008] Preferably, the waiting time for the second immunization is two weeks later, and the time for the third immunization is two weeks after the second immunization.

[0009] Preferably, the spleen is taken in a sterile environment during step 6.

[0010] Preferably, the cell suspension is prepared by collecting spleen cells by washing the spleen with culture medium, grinding the spleen cells on a stainless steel screen, and then separating, washing, and counting the spleen cells by centrifugation.

[0011] Preferably, in step 6, cell fusion includes centrifuging logarithmic growth phase myeloma cells, washing them with serum-free culture medium, counting and preparing a suspension, mixing it with the spleen cell suspension, with the ratio of myeloma cells to spleen cells being 1:5 to 10, and mixing them in a centrifuge tube.

[0012] Preferably, step 7 includes counting cells after staining with trypan basket, selective culturing with HT medium, seeding into 96-well plates for culture, and expanding and cryopreserving cells after positive detection.

[0013] Preferably, hybridoma cells are inoculated into BALB / c mice to collect ascites fluid. The ascites fluid contains 5-20 mg / ml of monoclonal antibody. The ascites fluid is then filtered through a filter membrane, and the purified anti-human hepatitis B surface antigen monoclonal antibody is obtained after elution.

[0014] The advantages of this invention compared with the prior art are as follows: In this invention, the anti-human hepatitis B surface antigen monoclonal antibody can be obtained by effectively removing impurities, resulting in a monoclonal antibody with high specificity, high purity, high titer, good stability, good uniformity, and strong reproducibility, which greatly improves the sensitivity and specificity of the reagent. At the same time, it adopts a derivation method based on mouse ascites preparation, which is low in cost, high in yield, and relatively simple in production process, and can achieve large-scale preparation and use in a short period of time. Detailed Implementation

[0015] The present invention will now be described in further detail.

[0016] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0017] Therefore, this invention is intended to cover such modifications and variations falling within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the invention are disclosed in or will be apparent from the following detailed description. It will be understood by those skilled in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0019] The terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may expressly or implicitly include at least one of that feature. In the description of the invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified. In the description of the present invention, "a number" means at least one, such as one, two, etc., unless otherwise expressly specified.

[0020] Unless otherwise shown or indicated in the operational embodiments, all figures used to represent the amounts, physicochemical properties, etc., of ingredients in the specification and claims are to be understood to be adjusted by the term "about" in all cases. For example, therefore, unless stated to the contrary, the numerical parameters listed in the foregoing specification and appended claims are approximations, and those skilled in the art can appropriately modify these approximations to obtain the desired characteristics by utilizing the teachings disclosed herein. The use of numerical ranges indicated by endpoints includes all numbers within that range and any range within that range; for example, 1 to 5 includes 1, 1.1, 1.3, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.

[0021] The preparation method of anti-human hepatitis B surface antigen monoclonal antibody, as shown, includes the following steps: Step 1: Extract human hepatitis B surface antigen HBsAg; Step 2: Select experimental animals, specifically mice; Step 3: Immunize mice by injecting purified HBsAg antigen subcutaneously and intraperitoneally; Step 4: Perform a second and third immunization using the same dose of antigen and the same method; Step 5: Perform a booster immunization using the same dose of antigen and the same method before cell fusion; Step 6: Three days after the booster immunization, remove the spleen from the mice to prepare a cell suspension, prepare myeloma cells, and fuse the cell suspension with myeloma cells to form hybridoma cells; Step 7: Screen the hybridoma cells and culture clones the screened hybridoma cells; Step 8: Prepare ascites fluid and purify the ascites fluid to obtain monoclonal antibodies.

[0022] The experimental animals were 8-12 week old BALB / c mice. The second immunization was performed two weeks later, and the third immunization was performed two weeks after the second immunization. Spleens were harvested in a sterile environment. To prepare the cell suspension, the spleen was washed with culture medium and then ground on a stainless steel sieve to collect spleen cells. The collected cells were separated by centrifugation, washed, and counted to prepare a cell suspension. Specifically, three days after the last booster immunization, the mice were euthanized by cervical sac, and the spleen was aseptically harvested. The spleen was washed once with culture medium, ground on a 400-mesh stainless steel sieve, and the spleen cells were collected. The spleen was centrifuged at 1200 rpm for 10 minutes, washed twice, and the spleen cells were counted to prepare a cell suspension.

[0023] In step 6, cell fusion includes centrifuging logarithmic growth phase myeloma cells, washing them with serum-free culture medium, counting and preparing a suspension, and mixing them with the spleen cell suspension. The ratio of myeloma cells to spleen cells is 1:5-10. The mixture is prepared by centrifuging at 1200 rpm for 10 minutes. Discard the supernatant, aspirate any remaining liquid, gently tap the bottom of the tube, and place the centrifuge tube in a 37°C water bath. While gently shaking, add 1 ml of 50% PEG at 37°C over 1 minute, allowing it to stand for 1 minute. Then add 1 ml of serum-free culture medium at 37°C over 1 minute, followed by 5 ml over 2 minutes, then 15 ml over 5 minutes, until the volume reaches 0.50 ml. Shake continuously while adding to ensure thorough dilution and terminate the PEG reaction. For hybridoma screening, centrifuge at 800 rpm for 10 minutes, discard the supernatant, and resuspend the cells in HAT selection medium containing 20% ​​fetal bovine serum. Add 100 μL to each well of a 96-well plate containing feeder cells, and add the remaining cells to each well of a 24-well plate (1-2 ml per well). Incubate at 37°C in a 5% CO2 incubator, changing half of the culture medium every 3 days.

[0024] Ascites preparation involves first injecting 0.5 ml of ipristane or liquid paraffin into BaLb / c mice intraperitoneally. One to two weeks later, 1 × 10⁸ hybridoma cells are injected intraperitoneally. Ascites will be produced 7 to 10 days after cell inoculation. The animals' health status and signs of ascites are closely observed. When ascites is as abundant as possible and the mice are close to death, the mice are sacrificed, and the ascites is aspirated into a test tube using a dropper. Generally, 1 to 10 ml of ascites can be obtained from one mouse. Ascites fluid can also be extracted using a syringe, and can be collected repeatedly several times. The ascites fluid is then filtered through a filter membrane. After pretreatment, it is filtered again to obtain purified anti-human hepatitis B surface antigen monoclonal antibody. Specifically, the antibody is filtered through a 0.45-micron filter membrane, desalted using a dextran gel G25 desalting column, equilibrated and eluted using phosphate buffer, and the protein solution is collected. The protein solution is then loaded onto a Protein A / G affinity column, and impurities are eluted from the Protein A / G affinity column using phosphate buffer. Finally, the antibody is eluted from the Protein A / G affinity column using glycine-hydrochloric acid solution to obtain the antibody solution.

[0025] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0026] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0027] The present invention and its embodiments have been described above, and such description is not restrictive. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A method for preparing monoclonal antibodies against human hepatitis B surface antigen, characterized in that: Includes the following steps: Step 1: Extract human hepatitis B surface antigen (HBsAg); Step 2: Select experimental animals; the experimental animals are mice. Step 3: Immunize the mice by injecting purified antigen HBsAg into the subcutaneous and peritoneal cavity. Step 4: Continue with the same dose of antigen and the same method for the second and third immunizations; Step 5: Before cell fusion, perform booster immunization using the same dose of antigen and the same method; Step 6: Three days after booster immunization, the spleen of the mouse was removed to prepare a cell suspension, myeloma cells were prepared, and the cell suspension was fused with myeloma cells to form hybridoma cells. Step 7: Screen hybridoma cells and culture clones of the screened hybridoma cells; Step 8: Prepare ascites fluid and purify the ascites fluid to obtain monoclonal antibodies.

2. The method for preparing anti-human hepatitis B surface antigen monoclonal antibody according to claim 1, characterized in that: The experimental animals used in step 2 are 8-12 week old BALB / C mice.

3. The method for preparing anti-human hepatitis B surface antigen monoclonal antibody according to claim 1, characterized in that: The waiting time for the second immunization is two weeks later, and the time for the third immunization is two weeks after the second immunization.

4. The method for preparing anti-human hepatitis B surface antigen monoclonal antibody according to claim 1, characterized in that: In step 6, the spleen is harvested in a sterile environment.

5. The method for preparing anti-human hepatitis B surface antigen monoclonal antibody according to claim 4, characterized in that: The cell suspension is prepared by collecting spleen cells by washing the spleen with culture medium, grinding the spleen cells on a stainless steel screen, and then separating, washing, and counting the spleen cells.

6. The method for preparing monoclonal antibody against human hepatitis B surface antigen according to claim 1, characterized in that: In step 6, cell fusion includes centrifuging myeloma cells in the logarithmic growth phase, washing them with serum-free culture medium, counting and preparing them into a suspension, and mixing them with the cell suspension prepared from the spleen. The ratio of myeloma cells to spleen cells is 1:5-10. The mixture is then centrifuged to obtain the final product.

7. The method for preparing anti-human hepatitis B surface antigen monoclonal antibody according to claim 1, characterized in that: Step 7 includes counting cells after staining with trypan basket, selective culturing with HT medium, seeding into 96-well plates for culture, and expanding and cryopreserving cells after positive detection.

8. The method for preparing monoclonal antibody against human hepatitis B surface antigen according to claim 1, characterized in that: Hybridoma cells were inoculated into BALB / c mice to collect ascites fluid. The ascites fluid contained 5-20 mg / ml of monoclonal antibody. The ascites fluid was filtered through a filter membrane, and the purified anti-human hepatitis B surface antigen monoclonal antibody was obtained after elution.