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(1) Fusion: Before cell hybridization, it is essential to prepare spleen B cell suspension and mouse myeloma cells (such as the SP2/0-Ag14 cell line) separately. The spleen from an immunized mouse is processed under aseptic conditions, and the B cells are suspended in a serum-free medium, typically RPMI 1640. The serum is removed three times to ensure purity. Meanwhile, SP2/0 cells are cultured in medium containing 10% fetal bovine or calf serum and refreshed daily to maintain vigorous growth. Both cell types are washed with RPMI 1640 and combined in the same tube. A 50% polyethylene glycol (PEG), with a molecular weight of 1000–1500, is used as the fusion agent and incubated at 37°C for 1–2 minutes. Afterward, the PEG is gradually diluted with RPMI 1640, removed, and the cells are dispersed into HAT selection plates. Electrofusion can also be used for monoclonal antibody production, though it requires specialized equipment and has lower cell numbers per fusion, limiting its widespread use. The optimal ratio of spleen cells to myeloma cells during fusion is usually between 5:1 and 10:1, with approximately 10^5 to 10^6 cells per fusion. The fused cells are then cultured in HAT medium (RPMI 1640 with 10–20% fetal bovine or calf serum and HAT) in 40- or 96-well plates at 37°C under 5% CO₂. Only the hybridoma cells formed by the fusion of spleen B cells and myeloma cells can grow in HAT medium, while other fused cells and unfused cells cannot survive.
In the culture process after fusion, feeder cells play a crucial role in supporting hybridoma growth. These can be peritoneal or thoracic-abdominal cells from the same species. Peritoneal macrophages help remove dead cell debris, creating a cleaner environment. Additionally, cytokines or active substances secreted by feeder cells promote hybridoma cell proliferation. Commercial "hybridoma growth factors" can also replace feeder cells in some cases.
(2) Screening of Positive Hybridoma Cells and Cloning: After about 10–14 days of culture, visible cell colonies (clones) begin to form. Antibody activity is tested after several media changes using HT medium. Common screening methods include ELISA and agglutination tests. ELISA is typically used for soluble antigens, while agglutination tests are more suitable for surface antigens like cells or bacteria. Other techniques such as Dot-ELISA, immunoblotting, and immunofluorescence can also be employed for hybridoma cell screening.
The process of isolating individual clones from a mixed population involves monoclonalization. The most widely used method is limited dilution, where the cell suspension is diluted and seeded into a culture plate so that most wells contain only one cell. To ensure that the resulting antibody-secreting cells come from a single parent cell, the cloning process may be repeated, known as subclonization. Another method used is fluorescence-activated cell sorting (FACS), which allows for more precise and efficient isolation of hybridoma clones.
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