Hybrid Cell Formation by Targeted Electrofusion

产生永生抗体的杂交瘤是由产生抗体的脾细胞与骨髓瘤细胞融合产生的。广泛使用的化学融合程序是时间密集的，需要大量有价值的抗原，并提供所需杂交瘤的相对较低的产量。传统的化学融合导致随机产生杂交瘤，其中大多数不能产生所需的抗体。这一建议是针对方法的发展，将增加抗原特异性细胞融合过程。骨髓瘤细胞首先被抗原（或半抗原）包裹。然后将这些标记的细胞与免疫小鼠的脾细胞孵育，然后它们与互补抗体结合。然后，两个细胞之间的融合可以通过化学或电融合来启动。一期项目结果表明：1)用半抗原或抗原标记骨髓瘤细胞表面是可能的；2)在适当的条件下，标记的细胞是有活力的；3)表面标记的骨髓瘤细胞与脾细胞孵育后，可以通过分子细胞选择找到互补淋巴细胞；4)靶向骨髓瘤。淋巴细胞二聚体可以融合产生杂交瘤。研究还表明，高速流式细胞术为监测这些过程提供了一种灵敏的方法。现在可行性已经被证明，第二阶段将为研究通过靶向电融合产生杂交瘤的最佳条件提供支持。PI希望利用靶向细胞电融合技术更有效地生产杂交瘤。传统的化学细胞融合导致随机产生杂交瘤，其中大多数不能产生所需的抗体。这里的PI打算利用抗原。b淋巴细胞的结合特性以增加细胞融合过程的特异性。这将克服定制杂交瘤生产商业化的一个重要限制。

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.