Biomedical Engineering Reference
In-Depth Information
only to a specifi c region of the antigen called an epitope (in the example above, the antigen con-
tains 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 pro-
tein surface would comprise fi ve to seven amino acid residues. Each specifi c antibody, which
recognizes a specifi c epitope, is produced by a specifi c B-lymphocyte. If one single antibody-
producing cell could be isolated and cultured
in vitro
, then it would be a source of monoclonal
(monospecifi c) 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 monospecifi c 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 specifi c
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.
13.3.1 Antibody screening: phage display technology
Phage display technology provides an extremely powerful modern way to generate a library of
(protein) ligands and, subsequently, screen these ligands for their ability to bind a selected target
molecule. The technique, as the name implies, employs fi lamentous phage (bacteriophage) that
replicate in
E. coli
.
The principle of phage display is presented in Figure 13.2. A library of genes (one of which codes
for the protein of interest) is fi rst generated/obtained. These genes are inserted (batch cloned) into
a phage library fused to a gene encoding one of the phage coat proteins (pIII, pIV or pVIII). The
phage are then incubated with
E. coli
, which facilitates phage replication. Expression of the fu-
sion gene product during replication and the subsequent incorporation of the fusion product into
the mature phage coat results in the gene product being 'presented' on the phage surface. The
entire phage library can then be screened in order to identify the one(s) coding for the protein of
interest. This is usually achieved by affi nity selection (biopanning). Biopanning entails passing
the library over immobilized target molecules, usually in immobilized column format. Only the
phage expressing the protein of desired specifi city should be retained in the immobilized col-
umn. The bound phage can subsequently be eluted, e.g. by reducing the pH of the elution buffer
or inclusion of a competitive ligand (usually free target molecules) in the buffer. Eluted phage
can then be repassed over the affi nity column in order to isolate those binding the immobilized
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