The basis of monoclonal antibody production by hybridoma technology

When antigen enters the body, it stimulates an immune response. A major element of this response entails activation of selected B-lymphocytes to produce antibodies capable of binding the antigen (the humoral immune response). The binding of antibody can reduce/inactivate the biological activity of the antigen (especially if it is a toxin, for example), and also marks the antigen for destruction by other elements of the immune system. Any given antibody will bind

Antibody producing B

In-vivo humoral immune response

In-vitro monoclonal antibody production

Antibody producing B

In-vivo humoral immune response

In-vitro monoclonal antibody production

Immortalized cell (hybridoma) producing antibodies of a single specificity

Figure 13.B1 Monoclonal antibody production

Immortalized cell (hybridoma) producing antibodies of a single specificity

Figure 13.B1 Monoclonal antibody production only to a specific region of the antigen called an epitope (in the example above, the antigen contains just three epitopes). Most antigens encountered naturally (e.g. proteins, viruses, bacteria, etc.) contain hundreds, if not thousands, of different epitopes. A typical epitope region on a protein surface would comprise five to seven amino acid residues. Each specific antibody, which recognizes a specific epitope, is produced by a specific B-lymphocyte. If one single antibody-producing cell could be isolated and cultured in vitro, then it would be a source of monoclonal (monospecific) antibody. However, B-lymphocytes die after a short time when cultured in vitro and, hence, are an impractical source of long-term antibody production (Figure 13.B1).

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.

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 selection medium. Individual hybridomas can be separated from each other by simple dilution and subsequently grown in culture, producing a clone. Individual clones can be screened to identify which ones produce murine (monoclonal) antibody that binds the antigen of interest. Appropriate clones are then selected and grown on a larger scale in order to produce biotechnologically useful quantities of antibody. Whereas many of the monoclonal antibodies approved in the 1980s and early 1990s were produced by such means, the majority of more recent approvals are engineered products produced by recombinant means, as described later.

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