General aspects of hybridoma production

The immune system is capable of mounting an immune response to an almost limitless number of different antigens. Within the antibody response to each simple or complex antigen, many clones with differing specificities, affinity and avidity can be activated to antibody production by the multiple antigenic determinants (epitopes) of an immunogen. Few, if any, true monoclonal antibody responses occur in responses to antigens, although in some responses a very restricted number of B cells clones may be engaged in antibody production.

In 1976 Köhler and Milstein described details of a powerful method in which B cells secreting antibody of one specificity could be fused with a continuously growing plasmacytoma. Clones of hybrid cells derived in this way, which express the immunoglobulin genes from both the normal antibody-forming B cell and the plasmacytoma, inherit the potential for unlimited growth from the continuously growing plasmacytoma parent and the generic material determining a given antibody specificity from both antibody-secreting parents. The term hybridoma was coined to describe the fusion product of a normal antibody-forming cell with a plasmacytoma; for this important scientific advance Köhler and Milstein received the Nobel Prize for Medicine or Physiology in 1984.

As illustrated in Figure 1, when an animal is immunized with a given antigen, the antibody products secreted by the array of B cell clones triggered by the various epitopes on the immunizing antigen subsequently appear as a heterogeneous mixture in the immune serum. However, by constructing hybridomas from a member of each epitope-specific clone and then cloning these hybrids, monoclonal antibodies can be separated from other monoclonals with specificities toward the same or other epitopes on the same antigen. It is then possible to isolate a number of monoclonal antibodies specific for different epitopes on the same molecule or immunogen. Such highly specific antibodies can be used to probe the structure and function of different parts of molecules and various cell types without need for immunoadsorptions, and at the same time a virtually unlimited supply of antibody can be obtained that is constant in its specificity and affinity over time.

Hypoxanthine, aminopterin, thymidine (HAT) metabolism and selection procedures

In fusion procedures between two cell populations the frequency of fusion is low (1 per 104 of the lymphoid cells). Therefore, in order to select against the neoplastic plasmacytoma or lymphoma lines used in B cell hybridoma formation, a strategy must be employed to kill the nonfused cells because they have the potential for continuous in vitro growth and would eventually overgrow the minority hybrid cells.

Salvage pathways

Hypoxanthine PRPP or -►

guanine HGPRT

Thymidine

Principal biosynthetic pathways for purines and pyrimidines

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