Hybrid-myeloma protocol

Figure 7. Flow chart of the hybrid-myeloma protocol incorporating the artificial antigens as immunizing antigens and screening aids.

Once inter-species fusion was shown to be possible, the next step was an attempt to fuse myelomas with normal B lymphocytes. Only in 1975 did Kohler and Milstein propose a protocol that led to the efficient production of hybrid cells for the secretion of mAbs with a predetermined specificity, which could be perpetuated in cell cultures, the so-called hybridomas (Kohler, 1981). 

Most current protocols use the approach of fusion with mutant myeloma cells according to a strategy in which the mutant cells are rescued from death by hybridization with lymphocytes in a medium otherwise lethal for this mutant (Littlefield, 1964 Ephrussi and Weiss, 1969) in which only the hybrids survive. Usually only a few hybrids produce the desired antibodies. 

A number of protocols have been described for the generation of mouse hybrid-omas and workers have their own particular protocol. We have used a standard procedure for the fusion of mouse and rat myelomas with considerable success over the last twenty years and this is described in Protocol 7. Basically, 10 lymphocytes are mixed with 2 x 10 mouse myeloma cells or 5 X 10 Y3 cells in a round-bottomed tube and pelleted by centrifugation. Then the ceUs are fused by the addition of 1 ml of 50% PEG 1500 and plated into HAT selection medium to allow the growth of the hybridomas generated. 

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