Antibody hybridoma fusion

Key Words Monoclonal antibodies cell fusion hybridoma immunization cell culture myeloma splenocytes. 

In production of the anti-RANTES monoclonal antibodies, after generation of the hybridoma fusions, plate ELISA was performed on 480 hybridomas. After repeated subcloning using limiting dilution analysis, four reproducibly positive hybridomas were characterized. 

Figure 4.33. Preparation of Monoclonal Antibodies. Hybridoma cells are formed by fusion of antibody-producing cells and myeloma cells. The hybrid cells are allowed to proliferate by growing them in selective medium. They are then screened to determine which ones produce antibody of the desired specificity. [After C. Milstein. Monoclonal antibodies. Copyright 1980 by Scientific American, Inc. All rights reserved.]...

Advantages of Monoclonal versus Polyclonal Antibodies, 406 When Should Monoclonal Antibodies be Made , 406 Immunization Prior to Fusion, 406 Hybridoma Fusion, 407 Screening Strategies, 407... 

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. 

Monoclonal antibodies are derived from a single, monospecific B cell clone. Monoclonal antibodies can be obtained from hybridoma cells that result from the fusion of antibody-producing B cells with immortal cells of a myeloma cell line. 

Monoclonal antibody technology entails isolation of such B-lymphocytes, with subsequent fusion of these cells with transformed (myeloma) cells. Many of the resultant hybrid cells retain immortal characteristics, while producing large quantities of the monospecific antibody. These hybridoma cells can be cultured long term to effectively produce an inexhaustible supply of the monoclonal antibody of choice. 

The basic principle for making the rabbit monoclonal antibody is the same as for mouse monoclonals. Rabbit fusion partner cells can fuse to rabbit B-cells to create the rabbit hybridoma cells. Hybridomas are then screened to select for clones with... 

Hybridoma Cell produced by the fusion of antibody-producing plasma cells with myeloma/carcinoma cells. The resultant hybrids have then the capacity to produce antibody (as determined by the properties of the plasma cells), and can be grown in continuous culture indefinitely owing to the immortality of the myeloma fusion partner. This technique enabled the first continuous supply of monoclonal antibodies to be produced. 

Polyclonal antibodies can react with many epitopes, whereas MAbs are restricted to one epitope on proteins that do not have repeating sequences.24 By definition, polyclonal immunoassays are generally much more sensitive but less specific than monoclonal assays. Bispecific or hybrid antibodies can be used to increase the affinity. Bispecific antibodies are formed by the fusion of two previously established hybridomas to produce antibodies displaying the binding characteristics of both of the antibodies in one molecule.25... 

The next development was the production of monoclonal antibodies (MAbs) in the mid-1970s. This uses hybridoma technology, which involves the fusion of antibody-producing B cells to immortal myeloma cells. Figure 4.4 shows the preparation of MAbs using hybridoma techniques. A more detailed discussion of biopharmaceuticals production is presented in Section 10.5. 

To obtain MABs, lymphocytes isolated from the spleen of immunized mice (1) are fused with mouse tumor cells (myeloma cells, 2). This is necessary because antibody-secreting lymphocytes in culture have a lifespan of only a few weeks. Fusion of lymphocytes with tumor cells gives rise to cell hybrids, known as hybridomas, which are potentially immortal. 

The assay principle should, however, be applicable to any target hapten, unlike assays based on a chemical modification. Cloning efficiency of the hybridoma-secreting anti-idiotype antibodies would be in a practical range, and much higher than that of anti-metatype antibodies. We (Kl) established four kinds of a-type and two kinds of /3-type anti-idiotype antibodies after three fusion experiments, each using spleen cells from one immunized mouse. Barnard et al. (Bl, B3, B4)... 

Spleen-derived B lymphocytes are then incubated with mouse myeloma cells in the presence of propylene glycol. This promotes fusion of the cells. The resultant immortalized antibody-producing hybridomas are subsequently selected from unfused cells by culture in a specific... 

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