Immortal antibody producing hybridomas are produced by the fusion of antibody producing spleen cells with myeloma cells. The widely used chemical fusion procedures are time intensive, require large amounts of valuable antigen and provide relatively low yields of the desired hybridomas. Conventional chemical fusion results in the random generation of hybridomas, most of which are not producing the desired antibodies. This proposal is directed towards the development of methods which will add antigen specificity to the cell fusion process. Myeloma cells are first coated with the antigen (or hapten). These labeled cells are then incubated with spleen cells from an immunized mouse, whereupon they bind to complementary antibody. Fusion between the two cells can then be intiated either chemically or by electrofusion. Results from the Phase I project have indicated that: 1) It is possible to label the surface of myelome cells with haptens or antigens, 2) Under proper conditions, the labeled cells are viable, 3) Upon incubation with spleen cells, surface labeled myeloma cells can find complementary lymphocytes by molecular cell selection, and 4) The targeted myeloma.lymphocyte dimers can be fused to yield hybridomas. It was also demonstrated that high speed flow cytometry provides a sensitive method for monitoring these processes. Now that feasibility has been demonstrated, Phase II would provide support to investigate optimal conditions for producing hybridomas via targeted electrofusion. The PI wishes to exploit the technique of targeted cell electrofusion for more efficient hybridoma production. Conventional chemical cell fusions result in random generation of hybridomas, most of which are not producing the desired antibody. The PI here proposes to exploit the antigen.binding properties of B.lymphocytes to increase the specificity of the cell fusion process. This would overcome an important limitation to the commercialization of custom hybridoma production.
