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864 Part 8: Food Safety and Food Allergensrecognition sites of the antibody, which interact with an epitope
on an antigen of interest, are located at the ends of the variable
heavy (VH) and variable light (VL) regions of the heavy and
light chains, respectively. They are commonly referred to as the
complementarity determining regions, or CDRs. Each arm of
an antibody can bind to one antigen, so one IgG molecule can
theoretically bind to two antigens.Antibody Production StrategiesThere are three main methods for generating antibodies that may
be used in biosensor-based platforms for quality evaluation, and
these are discussed in this section. Polyclonal antibodies are pro-
duced through the immunisation of animal hosts with a particular
antigen. The immunogen is typically administered in the pres-
ence of a suitable adjuvant, which elicits an immune response in
the host. Small molecules, such as toxins or haptens, may have to
be conjugated to larger carrier molecules, such as bovine serum
albumin (BSA), to enhance immunorecognition. Serum titres
are typically analysed by enzyme-linked immunosorbent assay
(ELISA) to quantify the host-based response. If this is deemed
to be suitable, blood samples are subsequently collected and the
antibodies generated are purified from the serum. Polyclonal an-
tibodies typically consist of a variety of different serotypes with
varying affinities/specificities towards the analyte in question.
Animals often used for polyclonal antibody generation include
guinea pigs, rabbits, goats, sheep and donkeys (Leenaars and
Hendriksen 2005).
The second method involves the use of hybridoma technology
to produce monoclonal antibodies (K ̈ohler and Milstein 1975,
Nelson et al. 2000, Hudson and Souriau 2003). These are gen-
erated by immunising an animal (typically, a mouse) with the
antigen of interest in the presence of a suitable adjuvant. Once
a sufficient immune response is detected by ELISA, the spleen,
bone marrow from long bones (femur and humerus) or primary
lymphoid organs (such as lymph nodes) are removed from the
sacrificed animal and the antibody-producing B-cells are har-
vested. These cells can then be fused to immortal myeloma cells
by using an electrical current or polyethylene glycol. The result-
ing hybrid cells (hybridomas), which secrete antibodies that are
directed towards the desired antigen, are then selected and cloned
out to ensure monoclonality. The advantage of this approach is
that there is a constant supply of the antibody that is required
for analysis. However, there is a significant cost involved in the
production and the screening of these antibodies.
Recombinant antibodies are the third form of antibodies that
are increasingly being used. They are often produced in bac-
terial strains such asEscherichia coliand are expressed in a
phage display format. Libraries, with the capacity to express a
large number of antibodies, are generated, and they are referred
to as naive, synthetic or immune depending on their mode of
production (Bradbury and Marks 2004). Immune libraries are
constructed though the administration of the immunogen of in-
terest to a suitable host (e.g. mouse, rabbit, chicken), which is
monitored for antibody production. Antibody-encoding nucleic
acid is then purified from lymphoid organs, such as the spleen,
and cloned into a phage or phagemid vector that, in turn, isVVH
VHCLVLCH1VHVHVLscFv FabFigure 45.5.Different antibody fragments available for biosensor-
based analysis. CH1–3refers to constant heavy region 1; VH,
variable heavy; VL, variable light; scFv, single-chain variable
fragment; Fab, fragment antigen binding.propagated inE. coli. Phage display libraries can subsequently
be screened against targets of interest by biopanning. This is
feasible since the phage express the active recombinant antibod-
ies on their surface and those that bind to the specific antigen
immobilised on a capture surface can be differentiated from non-
binding antibodies, and characterised further to determine their
affinity for the target antigen. A key advantage with recombinant
antibodies is the ability to increase their affinity for an antigen of
interest through site-directed mutagenesis or other approaches
such as chain shuffling or error-prone PCR, which is not possi-
ble for monoclonal or polyclonal antibodies (Conroy et al. 2009,
O’Kennedy et al. 2010).
The main types of antibody fragments produced by phage
display are the fragment antigen binding (Fab) and single-chain
variable fragment (scFv), whose structures are shown in Fig-
ure 45.5. The presence of the constant regions in a Fab is thought
to aid in the stabilisation of the antibody variable regions, which
might not function efficiently when expressed in the monomeric
scFv format (R ̈othlisberger et al. 2005). It was previously shown
in our laboratory that the Fab antibody format is the most reliable
and sensitive for use in small molecule competition biosensor
assays involving haptens. The strict monovalency of this format
can lead to a significant enhancement in assay sensitivity in both
ELISA and competition SPR assays (Townsend et al. 2006).
The scFv is the most widely used antibody fragment. The
variable regions (VHand VL) of the antibody are linked by a
flexible peptide linker. The most frequently used are based on
glycine-serine repeat structures, with the length of the linker
related to the intended valency of the molecule. When a short
linker is used, the stability and folding of the scFv does not occur
properly. This is caused by the insufficient juxtaposing of the VH
and VLregions in the single chain for the monomer to function.
ScFvs selected in this format invariably form bivalent dimers,
or diabodies, which often have increased avidity for an antigen
over the monomeric forms typically observed when long-linker
systems are used (Holliger et al. 1993, Kortt et al. 1997, Atwell