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Test each well for antibody to antigen X

Test each well for antibody to antigen X

▲ EXPERIMENTAL FIGURE 6-38 Use of cell fusion and selection to obtain hybridomas producing monoclonal antibody to a specific protein. Step 1: Immortal myeloma cells that lack HGPRT, an enzyme required for growth on HAT selection medium, are fused with normal antibody-producing spleen cells from an animal that was immunized with antigen X. The spleen cells can make HGPRT Step 2: When plated in HAT medium, the unfused cells do not grow; neither do the mutant myeloma cells, because they cannot make purines through an HGPRT-dependent metabolic "salvage" pathway (see Figure 6-41), and the spleen cells, because they have a limited life span in culture. Thus only fused cells formed from a myeloma cell and a spleen cell survive on HAT medium, proliferating into clones called hybridomas. Each hybridoma produces a single antibody. Step 3: Testing of individual clones identifies those that recognize antigen X. After a hybridoma that produces a desired antibody has been identified, the clone can be cultured to yield large amounts of that antibody.

one type of monoclonal antibody from blood is not feasible, in part because the concentration of any given antibody is quite low.

Because of their limited life span, primary cultures of normal B lymphocytes are of limited usefulness for the production of monoclonal antibody. Thus the first step in producing a monoclonal antibody is to generate immortal, antibody-producing cells. This immortality is achieved by fusing normal B lymphocytes from an immunized animal with transformed, immortal lymphocytes called myeloma cells. During cell fusion, the plasma membranes of two cells fuse together, allowing their cytosols and organelles to intermingle. Treatment with certain viral glycoproteins or the chemical polyethylene glycol promotes cell fusion. Some of the fused cells can undergo division and their nuclei eventually coalesce, producing viable hybrid cells with a single nucleus that contains chromosomes from both "parents." The fusion of two cells that are genetically different can yield a hybrid cell with novel characteristics. For instance, the fusion of a myeloma cell with a normal antibody-producing cell from a rat or mouse spleen yields a hybrid that proliferates into a clone called a hybridoma. Like myeloma cells, hybridoma cells grow rapidly and are immortal. Each hybridoma produces the monoclonal antibody encoded by its B-lymphocyte parent.

The second step in this procedure for producing monoclonal antibody is to separate, or select, the hybridoma cells from the unfused parental cells and the self-fused cells generated by the fusion reaction. This selection is usually performed by incubating the mixture of cells in a special culture medium, called selection medium, that permits the growth of only the hybridoma cells because of their novel characteristics. Such a selection is readily performed if the myeloma cells used for the fusion carry a mutation that blocks a metabolic pathway and renders them, but not their lymphocyte fusion partners that do not have the mutation, sensitive to killing by the selection medium. In the immortal hybrid cells, the functional gene from the lymphocyte can supply the gene product missing because of the mutation in the myeloma cell, and thus the hybridoma cells but not the myeloma cells, will be able to grow in the selection medium. Because the lymphocytes used in the fusion are not immortalized and do not divide rapidly, only the hybridoma cells will proliferate rapidly in the selection medium and can thus be readily isolated from the initial mixture of cells.

Figure 6-38 depicts the general procedure for generating and selecting hybridomas. In this case, normal B lymphocytes are fused with myeloma cells that cannot grow in HAT medium, the most common selection medium used in the production of hybridomas. Only the myeloma-lymphocyte hybrids can survive and grow for an extended period in HAT medium for reasons described shortly. Thus, this selection medium permits the separation of hybridoma cells from both types of parental cells and any self-fused cells. Finally, each selected hybridoma is then tested for the production of the desired antibody; any clone producing that antibody is then grown in large cultures, from which a substantial quantity of pure monoclonal antibody can be obtained.

Monoclonal antibodies are commonly employed in affinity chromatography to isolate and purify proteins from complex mixtures (see Figure 3-34c). They can also be used to label and thus locate a particular protein in specific cells of an organ and within cultured cells with the use of immunofluorescence microscopy techniques (see Figures 6-26a and 6-27) or in specific cell fractions with the use of immunoblotting (see Figure 3-35). Monoclonal antibodies also have become important diagnostic and therapeutic tools in medicine. For example, monoclonal antibodies that bind to and inactivate toxic proteins (toxins) secreted by bacterial pathogens are used to treat diseases caused by these pathogens. Other monoclonal antibodies are specific for cell-surface proteins expressed by certain types of tumor cells; chemical complexes of such monoclonal antibodies with toxic drugs or simply the antibodies themselves have been developed for cancer chemotherapy. I

HAT Medium Is Commonly Used to Isolate Hybrid Cells

The principles underlying HAT selection are important not only for understanding how hybridoma cells are isolated but also for understanding several other frequently used selection methods, including selection of the ES cells used in generating knockout mice (Chapter 9). HAT medium contains hy-poxanthine (a purine), aminopterin, and thymidine. Most animal cells can synthesize the purine and pyrimidine nu-cleotides from simpler carbon and nitrogen compounds (Figure 6-39, top). The folic acid antagonists amethopterin and aminopterin interfere with the donation of methyl and formyl groups by tetrahydrofolic acid in the early stages of the synthesis of glycine, purine nucleoside monophosphates, and thymidine monophosphate. These drugs are called an-tifolates because they block reactions of tetrahydrofolate, an active form of folic acid.

Many cells, however, are resistant to antifolates because they contain enzymes that can synthesize the necessary nu-cleotides from purine bases and thymidine (Figure 6-39, bottom). Two key enzymes in these nucleotide salvage pathways are thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HGPRT). Cells that produce these enzymes can grow on HAT medium, which supplies a

De novo synthesis of purine nucleotides PRPP (5-Phosphoribosyl-1-pyrophosphate)

^Blocked by antifolates

CHO from tetrahydrofolate

Nucleic acids

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