Myeloma antibody generation

Monoclonal antibodies generated by fusing mouse spleen cells to myeloma cells can be considered as a pure single reagent that is available in unlimited quantity. Drawbacks are high costs, clonal drift and often low affinity. 

Mammalian Cells Unlike microbial cells, mammalian cells do not continue to reproduce forever. Cancerous cells have lost this natural timing that leads to death after a few dozen generations and continue to multiply indefinitely. Hybridoma cells from the fusion of two mammalian lymphoid cells, one cancerous and the other normal, are important for mammalian cell culture. They produce monoclonal antibodies for research, for affinity methods for biological separations, and for analyses used in the diagnosis and treatment of some diseases. However, the frequency of fusion is low. If the unfused cells are not killed, the myelomas 1 overgrow the hybrid cells. The myelomas can be isolated when there is a defect in their production of enzymes involved in nucleotide synthesis. Mammahan cells can produce the necessary enzymes and thus so can the fused cells. When the cells are placed in a medium in which the enzymes are necessaiy for survival, the myelomas will not survive. The unfused normal cells will die because of their limited life span. Thus, after a period of time, the hybridomas will be the only cells left ahve. 

Figure 13.5 Outline of the production strategy of CEA-SCAN. The antibody-producing hybridoma cell line was originally obtained by standard methods of hybridoma generation. Spleen-derived murine B-lymphocytes were fused with murine myeloma calls. The resulting stable hybridomas were screened for the production of anti-CEA monoclonals. The clone chosen produces an IgG anti-CEA antibody. Note that the finished product outlined above is not radiolabelled. The freeze-dried antibody preparation (which has a shelf life of 2 years at 2-8 °C) is reconstituted immediately prior to its medical use. The reconstituting solution contains 99mTc, and is formulated to facilitate direct conjugation of the radiolabel to the antibody fragment...

As noted, one of the remaining challenges in obtaining human hybridoma cells for generahng human monoclonal antibodies is the lack of suitable human myeloma cell lines to generate stable hybrid cells that can be cloned and expanded indefinitely in culture. Many human hybridoma... 

Karpas, A., A. Dremucheva, and B.H. Czepulkowski, A human myeloma cell line suitable for the generation of human monoclonal antibodies. Proc Natl Acad Sci USA, 2001. 98(4) 1799-804. 

In the 1970s, the B-cell cancer myeloma was studied, and it was established that these cells produced a single type of antibody. A revolution started in 1975 in the area of generating antibodies when Kohler and Milstein succeeded in fusing murine... 

An immunogen induces antibodies from many B cell clones, producing a polyclonal antibody response. In contrast, the propagation of an isolated B cell clone produces an antibody of single specificity. However, the problem is that in tissue culture medium, B cells die within a few days of their isolation from, for example, a mouse spleen. To circumvent this problem, immortality can be conferred on B cells by means of viral transformation Epstein-Barr virus can be used. Alternatively, fusion to cancerous cells is carried out to generate hybrids or hybridomas. Generally, the former procedure is used to immortalize peripheral blood B cells and produce human monoclonal antibodies, while myeloma cells are used to produce murine monoclonal antibodies. 

We propose that this advanced technology has clear advantages over conventional methods for selective generation of novel monoclonal antibodies. It may also allow efficient production of stereo-specific monoclonal antibodies against native structure antigens with application of antigen-expressing myeloma cells. 

Antibody produced by a single clone of cells is a monoclonal antibody all the antibody molecules are identical and bind to the same antigenic site with identical binding affinities. Monoclonal antibodies can be generated in large amounts by creating a cell fusion (called a hybridoma) between an antibody-producing cell and a myeloma cell. 

Antibodies are expressed by hybridoma cells formed by cell fusion of sensitized animal or human B lymphocytes with myeloma cells, or they are generated by EBV (Epstein-Barr virus) transformation of sensitized B lymphocytes. Other heterologous expression systems such as bacteria, yeast, insect cells, and mammalian cells have also been used for expression of antibodies and their fragments. However, because of renaturation problems, glycosylation, and expression levels, mammalian cells are mostly used for the expression of monoclonal antibodies. More recently, technologies have been extensively developed for the expression of antibodies in transgenic animals and transgenic plants. 

Successful hybridoma production relies on the ability to (1) generate specific Rcells, (2) fuse them with a myeloma cell line, (3) identify the antibodies that are sought in culture supernatants, and (4) isolate and clone the... 

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