In the early 1970s, the field of antibody research was stymied by an inability to generate, isolate and purify single antibodies of a known specificity. On one hand, immortal myeloma cell lines were known to produce monoclonal antibodies or antibody fragments, though of unknown specificity (Henry Kunkel 1951). On the other hand, Norman Klinman and others had developed methods for cloning primary B cells that produced single antibodies of known specificity but were limited by low mAb yield and short cell lifespan. Cesar Milstein’s lab (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK) had been studying the origin of antibody diversity for a number of years and at the time was using the technique of cell:cell fusion to study the potential role of allelic exclusion in antibody expression in myeloma cells (Dick Cotton 1973). Georges Kohler joined Cesar Milstein’s lab as a postdoctoral fellow in 1974. Together they developed the idea of a hybrid cell resulting from a cell:cell fusion between an immortal myeloma cell and a short-lived antibody-producing B cell with a designed target specificity. These “hybridomas” could theoretically make monoclonal antibodies against any specific antigen. The scientists immunized mice with sheep red blood cells, a target known to elicit a strong antibody response in vivo, and fused the splenocytes with myeloma cells, creating the world’s first hybridomas. Each hybridoma possessed the immortal growth feature of the myeloma and the antibody-producing feature of the plasma cell. They screened for antibody target specificity by a plaque assay (lysis of sheep red blood cells) and identified a number of hybridomas producing target-specific antibodies. These hybridomas could be cloned, thus for the first time enabling production of large amounts of target-specific monoclonal antibodies.
The key figure and results from Kohler and Milstein’s 1975 Nature paper are included to the right:
“The cell line P3-X63Ag8 [myeloma] described above dies when exposed to HAT medium. Spleen cells from an immunised mouse also die in growth medium. When both cells are fused by Sendai virus and the resulting mixture is grown in HAT [hypoxanthine-aminopterin-thymidine] medium, surviving clones can be observed to grow and become established after a few weeks. We have used SRBC [sheep red blood cells] as immunogen, which enabled us, after culturing the fused lines, to determine the presence of specific antibody-producing cells by a plaque assay technique13 (Fig. 2a). The hybrid cells were cloned in soft agar14 and clones producing antibody were easily detected by an overlay of SRBC and complement (Fig. 2b). Individual clones were isolated and shown to retain their phenotype as almost all the clones of the derived purified line are capable of lysing SRBC (Fig. 2c).”
– Kohler and Milstein, Nature Vol. 256 August 7, 1975 p495-7.