Considerations for Immunizing Mice for Hybridoma Generation
The ClonaCell™-HY product line offers media for hybridoma development starting with myeloma cell culture, fusion with mouse B cells, HAT selection, expansion of fused cells, and clone stabilization. This workflow is preceded by mouse immunization for generating antibody-secreting cells that are later fused with myeloma cells. The immunization regimen should be carefully planned to generate robust hybridomas further downstream in the process. The immunization outcome depends on several factors including the strain of mouse, type and strength of the immunogen, and the immunization strategy and schedule, among others. Below are some considerations for immunizing mice for hybridoma generation.
- BALB/c splenocytes and parental myeloma cells of BALB/c mice are recommended for the following reasons: highly immune reactive, well-characterized, availability of myeloma cells from the same genetic strain. We recommend working quickly following the harvesting of the spleen and to perform the fusion as soon as possible (preferably within 1 hour). Cell viability and appearance can be assessed with the aid of a microscope and viability dye such as trypan blue. Splenocytes should appear smooth, shiny and round under a microscope. We don’t suggest using previously frozen splenocytes.
Type of Immunogens
- Immunogens such as purified proteins, carbohydrates, cell lines, or DNA/plasmids are used to elicit an immune response in mice for the development of hybridomas. Different immunogens can elicit stronger/faster responses than others and can influence class switching.
- Adjuvants can play an important role in the strength and type of antibody response elicited during immunization and can increase response to a weak immunogen. Commonly used adjuvants include alum, saponin derivatives and Freund’s adjuvant among others.
- Immunizations can be done by various routes including intra-peritoneal (IP), intravenous (IV), subcutaneous, and intranasal. In-house experience shows that IP immunizations favour stronger responses in the spleen. Other immunization routes may increase the response in lymph nodes or bone marrow.
- An immunization strategy and a proper immunization schedule should be planned ahead of time. After deciding on the type of immunogen, the amount to inject (usually 25-100 μg per injection), and the adjuvant, the user needs to set up an inoculation schedule. These can be longer regimes with boosts every 2-4 weeks for 2-3 months or rapid regimes, with multiple boosts per week for one month. A final antigen only (no adjuvant) injection should be administered a few days before harvesting the tissues for fusions.
- ClonaCell™-HY Products can be used for fusions of splenocytes, bone marrow, and lymph nodes from the immunized mouse. The preferred tissue type is spleen.
- Test bleeds collected between injections can help track the progress of the antibody response. Features such as class-switching can also be observed by assaying the sera in an ELISA. The test bleeds can help determine when to harvest the lymphoid organs for antibody-secreting B cells.
Cell Isolation Versus Whole Spleen
- We found that using the EasySep™ Mouse CD138 Positive Selection Kit gives a greater proportion of antigen-specific hybridomas relative to the use of total splenocytes for fusions.
How to Generate Hybridomas Using Semi-Solid Cloning
Watch a video outlining the steps in selection and growth of mouse hybridomas using ClonaCell™-HY.
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