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(1) Fusion: Prior to cell hybridization, it's essential to prepare a suspension of spleen B cells and mouse myeloma cells, such as the SP2/0-Ag14 cell line. The spleen from an immunized mouse is processed under sterile conditions, and B cells are suspended in a serum-free medium—typically RPMI 1640. The mouse serum is removed three times during this process. Meanwhile, SP2/0 cells are cultured in medium containing 10% fetal bovine or calf serum and refreshed daily to ensure vigorous growth during the logarithmic phase. Both cell types are then washed with RPMI 1640 and combined in the same tube. A 50% polyethylene glycol (PEG), with a molecular weight between 1000 and 1500, is used as a fusion agent and incubated at 37°C for 1–2 minutes. Afterward, the PEG is gradually diluted with RPMI 1640, removed, and the fused cells are dispersed into HAT selection plates. Alternatively, electrofusion can be used, though it requires specialized equipment and yields fewer cells per fusion, limiting its widespread use.
The optimal ratio of spleen B cells to myeloma cells during fusion is typically 5:1 to 10:1, with about 10^5 to 10^6 cells per fusion. The resulting hybrid cells are cultured in HAT medium (RPMI 1640 with 10–20% fetal bovine or calf serum and HAT) on 40- or 96-well plates at 37°C under 5% CO₂. Only hybridoma cells formed by the fusion of spleen B cells and myeloma cells will survive in HAT medium, while unfused cells and other types of fused cells cannot grow.
During the post-fusion culture, feeder cells can enhance hybridoma growth. These can be peritoneal or thoracic-abdominal cells from the same species. Peritoneal macrophages help remove dead cell debris, keeping the culture environment clean. Additionally, cytokines and bioactive substances secreted by feeder cells support hybridoma proliferation. Commercial "hybridoma growth factors" can also be used instead of traditional feeder cells.
(2) Screening of Positive Hybridoma Cells and Clonal Expansion: After approximately 10–14 days of culture, visible cell colonies (clones) begin to form. Antibody activity is tested after several medium changes using HT medium. Common screening methods include ELISA and agglutination tests. ELISA is preferred for soluble antigens, while agglutination is more suitable for surface antigens like cells or bacteria. Additional techniques such as Dot-ELISA, immunoblotting, and immunofluorescence can also be employed for hybridoma screening.
Clonal expansion involves isolating individual clones from a mixed population. The most common method is limited dilution, where the cell suspension is diluted and seeded onto a culture plate. This ensures that most wells contain only one cell. To confirm monoclonality, the process may be repeated—known as subcloning. Another technique used is fluorescence-activated cell sorting (FACS), which allows for precise isolation of single-cell clones.
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