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Once a good immune response has developed in an animal and an appropriate screening procedure has been developed, the construction of hybridomas is ready to begin. Polyethylene glycol (PEG) is the fusing agent of choice for hybridoma production, allowing the rapid and manageable fusion of mammalian cells. PEG fuses the plasma membranes of adjacent myeloma and/or antibody-secreting cells, forming a single cell with two or more nuclei. This heterokaryon retains these nuclei until the nuclear membranes dissolve before mitosis. In this protocol, antibody-secreting cells are isolated from the appropriate lymphoid tissue (mouse spleen and lymph nodes), mixed with myeloma cells, centrifuged to generate good cell-to-cell contacts, and fused with PEG. The fused cells are then diluted into selective medium and plated in multiwell tissue culture dishes. Beginning ∼1 wk later, samples of the tissue culture supernatants are removed from wells that contain growing hybridomas and tested for the presence of the appropriate antibodies. Cells from positive wells are grown, single-cell-cloned, and frozen. A procedure for screening batches of PEG for efficacy before hybridoma fusion is also included.

Polyethylene Glycol Fusion for Hybridoma Production