Myelomas and Monoclonal Antibodies
The tremendous diversity in antibody specificities present in the serum of an animal precludes any simple study of homogeneous antibody. Although purifications based on binding to an antigen might be at-tempted for obtaining antibody with one specificity, the resultant anti-bodies would still be highly heterogeneous, for many different antibodies normally are induced by a single antigen. Even a molecule as simple as phosphocholine can induce synthesis of hundreds of different antibodies. Instead of pursuing the purification of a single antibody out of the thousands that are induced, researchers have made use of a cancer of the immune system to obtain homogeneous antibod-ies.
A lymphocyte with unregulated cell division generates a cancer called myeloma. The serum of patients with myeloma contains high concen-trations of one single antibody. Myelomas can also be induced in mice, and the cancerous cells can be transferred from mouse to mouse. The ascites fluid collected from such mice is a solution of almost pure, homogeneous antibody. A particular myeloma can be indefinitely propagated, and its corresponding antibody can be easily purified and studied.
The major problem with the use of myelomas is that their antigens are unknown. Of course, it is possible to screen hundreds of myeloma antibodies against thousands of potential antigens to find a molecule the antibodies bind to, but this does not prove to be highly successful. It would be ideal to be able to induce a myeloma for any desired antigen. This can be done. Milstein and Kohler discovered that the immortality of a myeloma cell line can be combined with the selectable antigen specificity of antibodies that are obtained from B cells of immunized animals. If a variant myeloma line has been used that itself does not produce antibody, the result of the fusion is a cell line that synthesizes a single type of antibody. This has the desired binding specificity. The cell line can be grown in vitro or injected into mice, giving them myeloma but providing a convenient source of large quantities of their antibody product. The antibodies produced by these means are called monoclonal because they derive from a single cell or clone.
Figure 20.2 Scheme for the production of cell lines synthesizing monoclonalantibodies. Neither of the parental cell types can grow in the hypoxanthine aminopterin medium.
Mouse cell lines synthesizing monoclonal antibodies are isolated by fusing myeloma cells with lymphocytes extracted from the spleen of a mouse about five days after its immunization with the desired antigen (Fig. 20.2). The cell fusion is accomplished merely by mixing the two cell types in the presence of polyethylene glycol.
This agent induces fusion of the cell membranes; after an interval of unstable growth in which a number of chromosomes are lost, relatively stable chromosome numbers, called karyotypes, are established in the population. To re-duce the background deriving from growth of unfused parental cell types, a genetic selection against both of the parental cell types is used so that only a fusion product can grow. The fused cells are then diluted to a concentration of a few cells per well of a culture tray, and after they have had time to grow and secrete antibody to the growth medium, the individual cultures from the wells are screened for the presence of antibody with the desired specificity.
Monoclonal antibodies find wide use in diagnostic and therapeutic medicine and in many types of basic research. In addition to providing an almost limitless supply of antibody with unchanging characteristics, monoclonal technology permits antibodies to be made against materials that cannot be completely purified. For example, monoclonal antibod-ies can be made against the cell walls of neurons from an animal. The neurons are dissected out, cell walls isolated and injected into a mouse, and the fusion of spleen cells to the myeloma line performed. Even unfractionated brain could be used for the initial injections. The anti-bodies synthesized by the fused cells can be screened for desired properties. Some of the resulting monoclonal antibodies have been found to bind to all cells, some bind only to neurons, and others bind to just a few neurons. Presumably neurons of the latter class are closely related to one another in lineage or function.
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