In Vitro Immunization Introduction for Hybridoma Generation

In vitro immunization is a crucial immunization strategy for antibody production from hybridoma by directly triggering B cell responses to target antigens. This method is more convenient and safer immunization strategy. More importantly, since in vivo immunization is applicable to antigens with strong immunogenicity and sufficient sources, it is not applicable to weakly in immunogenic or harmful antigens (such as causing immunosuppression). If human monoclonal antibodies are produced, in vivo immunization is virtually impossible. In vitro immunization can also bypass ethical restrictions and obtain pure human antibodies by using human peripheral blood mononuclear cells, promoting the research progress of antibody therapy.

Steps of In Vitro Immunization

In vitro immunization involves coculturing normal animal or human spleen cells (or lymph node cells, peripheral blood lymphocytes) with antigens in vitro under specific conditions, and then fusing them with myeloma cells to achieve the immune effect. Here, we will take mice as an example to explain how to perform in vitro immunization..

Spleens from 4- to 8-week-old BALB/c mice are harvested in a sterile environment and made into single-cell suspensions. The single cell suspension is washed 2-3 times with serum-free medium, then suspended in culture medium containing 10% calf serum. The appropriate amounts of antigen (0.5-5 ug/ml for soluble antigens, 105-106 cells /ml for cellular antigens) and a certain amount of BALB/C mouse thymocyte culture supernatant are added. The suspension cells are cultured at 37°C, 6% CO₂ for 3-5 days, and then the spleen cells were isolated and fused with myeloma cells.

In vitro and In vivo Combined Immunization Strategies

In vitro immunization can complement the deficiencies of in vivo immunization and can be applied to antigens with weak immunogenicity that require antibody preparation or that can inhibit the immune response of animals. Combining in vitro and in vivo immunization can be more effective in immunizing antigens and obtaining higher titers of antibodies. In the case of mice, the steps of combined in vivo and in vitro immunization are as follows:

In vivo immunization can be performed by intrasplenic method, intraperitoneal or subcutaneous multiple point injection 2-3 times. Boosting immunization before fusion is replaced by in vitro induction. That is, the spleens of immunized mice are taken by conventional methods to prepare spleen cell suspension. The cells are suspended in a complete culture medium and added with rHuIL-2, rHuIL-6, PWM, and specific immunogen to the final concentrations of 50 U/ml, 500 U/ml, 1: 200 and 500 mg/ml, respectively. After 5 days of incubation in a 5% CO₂ incubator at 37 °C, the cells are harvested for fusion.

Creative Biolabs has extensive experience in the field of in vitro immunization and is eager to share its knowledge of in vitro immunization with you. We sincerely wish you the best in your research.

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