A better cell line for making hybridomas secreting specific antibodies

Abstract

270 were found and we conclude that revertants constitute fewer than one in 5 x 10 4 of the TNP-specific hybridoma cells. The karyotypes of the TNP-specific cell lines provide evidence that these cells are hybrids of Sp2/0-Ag14 and mouse cells. Normal mouse cells and Sp2/0-Ag14 contain 40 and 73 chromosomes, respectively. The TNP-specific cell lines hyl.2, hy2.15, hy3.3 and hy5.19 contain approximately 100, 105, 106 and 99 chromosomes, respectively, and we suppose that fusion yielded hybrids that segregated better growing cells that had lost 7-14 chromosomes. Evidence has been presented that X63-Ag8 cells preferentially fuse with the dividing cells of the spleen 2 • 6 .1, which for the B-cell compartment consist mainly of lg-secreting, plaque-forming cells. Sp2/0-Ag14 seems to display the same preference. In the population of spleen cells used for this fusion, approximately 0.4% of the cells made indirect (IgG) TNP-specific plaques and 0.004% made direct (IgM) TNP plaques. Assuming that 1 % of the spleen cells are plaque forming cells 8 one would anticipate that about 40% of the hybridomas would make lgG with TNP specificity, as was observed. On the other hand, about 90% of the nonspecific plaque-forming cells of the spleen make lgM rather than IgG 8 , so that one would anticipate that among the nonspecific hybridomas, 90% would also make lgM, a figure that is consistent with our observation that all four nonspecific hybridomas examined made lgM. To obtain high-titre preparations of the TNP-specific lg, 10 6 TNP-specific hybridoma cells were injected intraperitoneally into BALB/c mice. All caused tumours and yielded ascites fluid with titres generally about 100 times higher than for the culture supernatants. The efficiency of fusion with Sp2/0-Ag14 has been variable. In our early experiments, such as the fusion described above, we obtained fewer hybrids than we ordinarily obtain with X63-Ag8. However, in more recent experiments, fusions with Sp2/0-Ag14 have often been more efficient, in particular in fusions with lipopolysaccharide stimulated spleen cells or with large spleen cells (F. Melchers, Clark and G.K., unpublished observations). We consider that Sp2/0-Ag14 can yield as many hybrids as X63-Ag8. Alternative methods exist for obtaining hybridomas that do not express the myeloma lg. It has been possible to isolate re-clones that have lost the expression of the nonspecific heavy or light chains 2 • This problem has been reduced by fusing with NSI-Ag 4/1, a derivative of X63 that makes the K but not the yl chain of the myeloma 2 • We anticipate that Sp2/0-Agl4 will prove yet a better cell line for generating hybridomas making truly monoclonal antibodies.