The ability to create an immortal cell line producing large quantities of a completely characterized and highly specific antibody, known as a monoclonal antibody, has revolutionized immunologic testing. Monoclonal antibodies are produced by the offspring (clones) of a single hybrid cell, the product of fusion of an antibody-producing plasma B cell and a malignant antibody-producing myeloma cell. One technique for the production of such a clone of cells, called hybridoma cells, is illustrated in Figure 9-2.
The process starts by immunizing a mouse with the antigen for which an antibody is to be produced. The animal responds by producing many antibodies to the epitope injected. The mouse's spleen, which contains antibody-producing plasma cells, is removed and emulsified so that antibody-producing cells can be separated and placed into individual wells of a microdilution tray. Because cells cannot remain viable in cell culture for very long, they must be fused together with cells that are able to survive and multiply in tissue culture, that is, the continuously propagating, or immortal cells, of multiple myeloma (a malignant tumor of antibody-producing plasma cells). The special myeloma tumor cells used for hybridoma production, however, possess a very important defect—they are deficient in
Capsule (hyaluronic acid) T, R proteins
Polysaccharide W-acetyl glucosamine W-acetyl galactosamine rhamnose, glucose, galactose (group-specific antigens)
Fimbriae (M protein)
Figure 9-1 Group A Streptococcus (Streptococcus pyogenes} contains many antigenic structural components and produces various antigenic enzymes, each of which may elicit a specific antibody response from the infected host.
the enzyme hypoxanthine phosphoribosyltransferase. This defect leads to their inability to survive in a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium). Antibody-producing spleen cells, however, possess the enzyme. Thus, fused hybridoma cells survive in the selective medium and can be recognized by their ability to grow indefinitely in the medium. Unfused antibody-producing lymphoid cells die after several multiplications in vitro because they are not immortal, and unfused myeloma cells die in the presence of the toxic enzyme substrates. The only surviving cells will be true hybrids. The growth medium supernatant from the microdilution tray wells in which the hybridoma cells are growing is then tested for the presence of the desired antibody. Many such cell lines are usually examined before a suitable antibody is found because it must be specific enough to bind only the type of antigen to be tested, but not so specific that it binds only the antigen from the particular strain with which the mouse was first immunized. When a good candidate antibody-producing cell is found, the hybridoma cells are either grown in cell culture in vitro or they are reinjected into the peritoneal cavities of many mice, where the cells multiply and produce large quantities of antibody in the ascitic (peritoneal) fluid that is formed. Ascitic fluid can be removed from mice many times over the animals' lifetimes, and antibody molecules will be identical to the original done.
Polydonal and monodonal antibodies are both used in commerdal systems to detert infectious agents.
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