Hybridomas generation

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...

The vast majority of hybridomas generated in laboratories are destined to be discarded because they will not have the desired qualities of antibody specificity, growth characteristics, or cloning ability required. In most cases, it is more practical to derive a new cell line rather than try to continue with one that is less than ideal. It is very important to have in mind the qualities of the cell line that are required along with the characteristics of the antibody that are needed before embarking on hybridoma production. 

A number of protocols have been described for the generation of mouse hybrid-omas and workers have their own particular protocol. We have used a standard procedure for the fusion of mouse and rat myelomas with considerable success over the last twenty years and this is described in Protocol 7. Basically, 10 lymphocytes are mixed with 2 x 10 mouse myeloma cells or 5 X 10 Y3 cells in a round-bottomed tube and pelleted by centrifugation. Then the ceUs are fused by the addition of 1 ml of 50% PEG 1500 and plated into HAT selection medium to allow the growth of the hybridomas generated. 

Keitma, E. W. M. Wolbers, F Vermes, I. van den Berg, A. On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation. Electrophoresis 2011, 32, 3138-3146. 

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. 

Generation of antibodies that can recognize and bind to specific viruses is straightforward. A sample of live or attenuated virus, or a purified component of the viral caspid, can be injected into animals to stimulate polyclonal antibody production (or to facilitate monoclonal antibody production by hybridoma technology). Harvested antibodies are then employed to develop specific immunoassays that can be used to screen test samples routinely for the presence of that specific virus. Immunoassays capable of detecting a wide range of viruses are available commercially. The sensitivity, ease, speed and relative inexpensiveness of these assays render them particularly attractive. 

The invention of B cell hybridoma technology (K3) has allowed the generation of various kinds of useful antibodies, even very minor or rare antibody species elicited in serum by the conventional procedure, as a pure immunochemical reagent in almost unlimited amounts. Among such new-generation monoclonal antibodies, anti-idiotype antibodies and anti-immune complex (anti-metatype) antibodies have been successfully introduced as key reagents enabling noncompetitive hapten immunoassays (Fig. 11). 

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... 

Fusion with the cells compensates for this deficiency. When fused and unfused cells are incubated in the presence of the folic acid antagonist aminopterin, the de novo synthesis of purines and pyrimidines for DNA is blocked. Cells deficient in HGPRT die, whereas hybrid cells are able to bypass aminopterin blockage by metabolism of hypoxanthine and thymidine added to the medium. In the generation of mouse hybridomas, an number of myelomas deficient in HGPRT are available, all originating from MOPC 21, a spontaneous myeloma from the BALB/c mouse strain. 

A number of protocols will be described here that have been used successfully with both rat (Y3 and IR983F) and mouse (SP2/0) myelomas to generate MAbs to cellular proteins, recombinant proteins, or peptides based on cDNA sequences. Successful hybridoma production relies on the ability to ... 

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