Mouse Monoclonal Antibody Preparation

1. Basic Principle

Monoclonal antibodies are highly homogeneous antibodies produced from a single B cell clone that target only a specific epitope. Hybridoma antibody technology is based on cell fusion technology, which fuses sensitized B cells with the ability to secrete specific antibodies and myeloma cells with unlimited reproduction ability into B cell hybridoma.

Mouse myeloma cells, which can be cultured and proliferated in vitro, were fused with pure mouse B cells immunized by antigen to form hybrid cell lines, which not only have the characteristics of infinite proliferation of tumor cells in vitro, but also have the characteristics of synthesis and secretion of specific antibodies by antibody-forming cells. The hybridoma can be cultured as a single cell to form a single cell line, that is, monoclone. Using the method of culture or intraperitoneal inoculation of mice, a large number of high-concentration, very uniform antibodies can be obtained, whose structure, amino acid sequence, specificity, etc., are consistent, and in the culture process, as long as there is no variation, the antibodies secreted at different times can maintain the same structure and function. This monoclonal antibody cannot be obtained by any other method.

2. Preparation Process

2.1 Immunity in Laboratory Animals

（1）First Emulsification: Take the antigen (each 150-200µg) and the same volume of the full adjuvant of the Frosted mixture, through the medical tee tube and syringe for repeated pushing emulsification, the general requirements to achieve the emulsion of the mixture drops to the surface of the water can maintain the drop shape, does not rapidly spread until.

（2）Primary immune injection: Multi-point injection method was adopted, and the injection site was preferentially selected under the limbs, under the armpit, and then on the back.

（3）Follow-up immunization: The antigens (80-100µg each) were mixed with the same volume of Freund's incomplete adjuvant, and the emulsification process and immune injection process were exactly the same as the first immunization.

（4）After the four vaccines, the blood was collected from the mice with the tail severed, and the serum titer was determined by indirect ELISA using the serum as the primary antibody. When the serum dilution ratio was 12800, OD value ≥1 could be judged to meet the fusion criteria.

（5）Dash immunity: The antigen stock solution (200-250µg each) was taken and injected directly into the abdomen, and cell fusion was performed 3-5 days later.

2.2 Cell Fusion

（1）SP2/0 cells and immune mouse spleen cells were suspended with 1640 incomplete medium, and counted with blood cell counting plate, respectively. Spleen cells: SP2/0 cells =1:3 were mixed in a 50ml aseptic centrifuge tube, and 1640 incomplete medium was added to 40ml after full mixing.

（2）Centrifuge 1,500rpm for 5min. (At this time, prepare 37℃ warm water for the incubation of the cell fusion centrifuge tube.)

（3）After centrifugation, discard the serum and tube wall liquid (can be dried by sterilized absorbent paper), gently knock SP2/0 cells and immune mouse cells mixed precipitation to loosen them, and immerse the bottom of the centrifugation tube in 37℃ warm water.

（4）Take 1mL of the incubated fusion agent PEG1450 out of the incubator and evenly drop it into the cell mixed precipitation within 60s (drop and turn the centrifuge tube).

（5）Let it rest for 45s, take out the preheated 1640 incomplete medium in the 37℃ temperature tank, take 1mL, drop evenly into the precipitation to dilute PEG fusion agent (turn the centrifuge tube while adding drops), take another 1mL, drop evenly within 30s, and then slowly drop all the remaining 45mL of medium.

2.3 Assay of Serum Titer in Immunized Mice

（1）After four immunizations, the mice underwent tail amputation and blood collection.

（2）The collected whole blood of mice was placed at 37℃ for 1 h, then at 4℃ for 1 hour, or overnight at 4℃, then centrifuged at 3000-4000 rpm for 5-10 min, and the supernatant was taken and placed in a new clean EP tube, which was the serum of immune mice.

（3）The serum of one mouse was randomly selected to determine the optimal coating concentration by checkerboard method. The concentration gradient of antigen coating was usually set at 0.25-10μg/ml, and the dilution of serum was usually 400 or 500 times as the initial dilution.

（4）OD_450nm was determined by indirect ELISA, and the results were made into a statistical line chart. By observing the starting point of the line chart, it was determined that when the OD_450nm value of the starting point no longer increased significantly with the increase of the coated antigen concentration, and the whole line showed a smooth downward trend, the coated concentration was the best coated concentration.

（5）The serum titer of other mice was determined with the optimal coated concentration. When the serum dilution was 12800 times and the OD_450nm value was ≥1.0, it could be judged to meet the fusion requirements.

3. Advantages and Limitations of Monoclonal Antibodies

The advantages of monoclonal antibodies: (1) Hybridoma can survive and pass through in vitro "permanently", as long as there is no genetic mutation of the cell line, it can continue to produce highly specific and highly uniform antibodies. (2) A large number of highly specific, uniform antibodies can be obtained from relatively impure antigens. (3) Because it is possible to obtain "unlimited" homogeneous antibodies, it is suitable for immunological analysis methods characterized by labeled antibodies, such as IRMA and ELISA. (4) Due to the high specificity and single biological function of monoclonal antibodies, they can be used for radioimmunoimaging and immune-directed therapy in vivo.

Limitations of monoclonal antibodies: (1) The inherent affinity and limited biological activity of monoclonal antibodies limit its range of applications. Since monoclonal antibodies cannot precipitate and agglutinate, many assays cannot be performed with monoclonal antibodies. (2) The reaction strength of monoclonal antibody is not as strong as that of polyclonal antibody. (3) The preparation technology is complex and time-consuming, so the price of monoclonal antibodies is also high.

4. Application of Monoclonal Antibodies

4.1 Testing Medical Diagnostic Reagents

As a diagnostic reagent in laboratory medicine, monoclonal antibody is widely used in enzyme-linked immunosorbent assay, radioimmunoassay, immunohistochemistry and flow cytometry because of its strong specificity, high purity and good uniformity. And the application of monoclonal antibodies has greatly promoted the development of commercial kits.

4.2 Purification of Protein

Monoclonal antibody is an important ligand in affinity chromatography. Monoclonal antibodies were adsorbed on an inert solid matrix (e.g. Speharose 2B, 4B, 6B, etc.) and prepared into chromatographic columns. When the sample flows through the chromatographic column, the antigen to be separated can bind specifically to the solid monoclonal antibody, and the other components cannot bind to it. After the chromatographic column is fully eluted, the ionic strength or pH of the eluent is changed, the antigen to be separated is separated from the antibody, and the antigen to be purified can be obtained by collecting the eluent.

4.3 Tumor Targeted Therapy and Radioimmunoimaging Techniques

The monoclonal antibody against a tumor antigen is connected with chemotherapy drugs or radiotherapy substances, and the drug or radiotherapy substance is carried to the target organ to kill the target cells directly by using the guiding effect of monoclonal antibody. In addition, radio-immune imaging can be performed by linking radiolabels with monoclonal antibodies and injecting them into patients to assist in tumor diagnosis. Monoclonal antibodies are mainly murine antibodies, and xenogenic animal serum can cause allergic reactions in humans. Therefore, the preparation of human-human monoclonal antibodies or humanized antibodies is more important, but no significant progress has been made in this regard.

KMD Bioscience has been working on antibody preparation for many years. Monoclonal antibodies are produced by monoclonal hybridoma cells that fuse activated B lymphocytes with myeloma cells. We have experienced antibody preparation technicians, accumulated sufficient antibody preparation experience and professional technology, with perfect antibody production equipment, can provide customers with different species of monoclonal antibody customized services.

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