In order to obtain an unlimited supply of a defined antibody to an antigen in the laboratory, all that is required is the isolation and immortalization of the relevant clone of B-lymphocytes from an animal. The clone could be isolated and grown under laboratory conditions by cell culture. In this way the cloned B-lymphocytes would continue to produce the antibody, which could be collected from the fluid supernatant bathing the cells in culture. However, most attempts to grow clones of B-lymphocytes in the laboratory have failed. What is required therefore is to develop some method by which antibody-producing B-lymphocytes can grow freely and well in laboratory culture.
In the early 1970's it was already possible to grow several types of cell under just such conditions. One type of cell that could be grown easily was a malignancy of B-lymphocytes known as myeloma or plasmacytoma. These cells have the ability to reproduce themselves indefinitely, unlike their normal non-cancerous counterparts. Furthermore such cells are clonogenic--that is single cells will grow up to form clones either in vitro or when injected into suitable animals. If the property of growth in vitro and clonogenicity from the myeloma cell line could be combined with the property of antibody manufacture from the B-lymphocyte, the possibility of immortalizing defined antibody production could be overcome (Fig. 1.3).
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results in an immortal antibody-producing hybridoma |
In 1975, Köhler and Milstein demonstrated that myeloma cells could be fused with B-lymphocytes and the resultant hybrids produced antibodies. The technical details and problems of doing this will be explained later. When the fusion process is complete the daughter cells are known as "hybridomas," i.e. hybrid cells which have inherited some characteristics from both parents and which grow immortally as malignant cells. The characteristics required, which are actively selected for, are immortality from the malignant myeloma cell and antibody production from the B-lymphocyte. Thus, hybridization techniques allow immunoglobulin-producing cells to be immortalized. However, they do not determine which lymphocyte is being immortalized. To achieve this Köhler and Milstein developed the ingenious technique of immunizing their experimental animal with the antigen of interest. A mouse was immunized by the injection of the antigen--sheep red blood cells in the first experiment--on several occasions. After several weeks the mouse's immune system would be switching on its own inherent clones of B-lymphocytes which were capable of producing antibodies that recognized the test antigen. The mouse lymphocytes were collected from its spleen were used in the fusion process with the myeloma line. In this way it was likely that many of the lymphocytes fused would be secreting the antibody against the antigen of interest. It is important to understand that even using techniques such as this, in which the animal is primed to produce specific antibodies, many lymphocytes will be fused which are producing irrelevant antibodies. When the successful hybridization of myeloma and B-lymphocyte takes place and a colony, or single clone, of hybridoma cells all producing one antibody is established, this antibody is referred to as a monoclonal.
Although the fusion techniques outlined above enable antibodies of defined specificity to be produced in endless quantities, it should be stressed right from the beginning that for every successful antibody which is produced many failed fusions or irrelevant monoclonals will be produced. Many hours of laboratory time are spent to produce a single useful MCA. Some of the reasons for this are obvious. First of all the definition of what is a "good" monoclonal antibody is arbitrary and depends upon what function is required of it by the investigator. For example, some antibodies may be extremely good at recognizing certain types of lymphocytes in pathological sections any may help the histopathologist in the diagnosis of difficult diseases. However, the staining of such sections may be successful only under certain stringent conditions, such as the use of fresh tissue, and so severely limit the practical application of the antibody to circumstances in which tissue can be collected and examined in a fresh condition. It is therefore important for an investigator to know what he requires of his antibody before deciding which antibodies are good or bad. Further, many antigens against which the investigator is attempting to raise monoclonal antibodies are only weakly immunogenic. The animal's immune system therefore responds poorly to the immunogen and so the incidence of suitable monoclonals is low, thus increasing the workload.
