If cells taken from various clones of normal antibody- producing and secreting lymphocytes could be grown in culture, it would be possible to have ready sources of various antibodies at one’s disposal.
Unfortunately, laboratory cultures of normal lymphocytes grow very slowly or die out quickly.
There is, however, an abnormal and cancerous form of lymphocyte called a myeloma cell that can be cultured in the laboratory. Myeloma tumors can be produced in experimental animals by intra peritoneal injection of mineral oil.
In 1975, C. Milstein and G. Koehler were able to fuse myeloma cells with normal B lymphocytes, thereby producing a hybrid cell that could be grown in culture. Moreover, the hybrid cell, called a hybridoma, produces and secretes antibodies characteristic of the clone from which the normal lymphocyte is taken. The procedure for producing a hybridoma is depicted in Figure 25-11. First, an immune response is induced in a normal animal by exposure to a specific antigen (either by injecting the purified antigen or by injecting bacteria or viruses).
Lymphocytes are then obtained from the animal’s spleen or other lymphoid tissue. Among the cells that are removed will be some from clones that were activated by exposure to the antigen (many, perhaps most, of the cells will be from clones not selected by the antigen).
The normal lymphocytes are then mixed with myeloma cells in a solution of polyethylene glycol. The myeloma cells to be used lack the enzyme hypoxanthine-quanine phosphoribosyl transferase (HGPRT), which catalyzes the synthesis of inosine monophosphate (IMP) and guano- sine monophosphate (GMP). Polyethylene glycol induces fusion of the two families of cells, thereby forming hybridomas.
Some of the hybridomas that are formed will be hybrids of myeloma cells and the antigen-activated normal lymphocytes. Of course, the polyethylene glycol solution will also contain a number of unfused myeloma cells and normal lymphocytes.
The un-fused lymphocytes will fail to grow (or grow so slowly that they produce insignificant numbers of progeny) when the cells are subcultured. If subculturing is carried out in a medium containing hypoxanthine, aminopterin, and thymidine (i.e., HAT medium), un-fused myeloma cells will also die out because they cannot produce HGPRT.
The hybridomas will proliferate because the lymphocyte nuclei contribute functional HGPRT genes to the heterokaryon. The particular hybridomas producing the desired antibody can be identified using a suitable assay procedure, and these can be selected and separately cloned. Thus, the end result is a culture of cells producing a single type of antibody—a monoclonal antibody. Such cultures can be maintained indefinitely and be used as a continuous source of antibody.