732 Part VII: Food Safety
and production levels are generally low. Within the
food industry, what is needed is a cheap supply of
large amounts of antibodies to perform high-
throughput screening for pathogens.
Recombinant antibodies fill this void since they
can be produced in reasonable quantities in short
periods of time from bacterial expression systems.
Handling, growth, and storage of the bacteria are
simpler and cheaper than the corresponding work
with hybridoma cells (Breitling and Dübel 1999).
Recombinant antibodies also have the advantage of
being clonal—one antibody fragment is produced
per bacterial colony. The use of recombinant anti-
bodies offers the opportunity to select binding anti-
bodies from naïve rather than immunized antibody
libraries, removing any ethical dilemmas related to
immunizing animal hosts with known pathogenic
organisms (Churchill et al. 2002) or sacrificing the
animals for their spleens. Finally, recombinant anti-
bodies can easily allow one to improve the binding
properties and affinities of an antibody through
mutagenesis and genetic engineering of the expres-
sion vectors and genes (Yau et al. 2003).
Recombinant antibodies come in a number of
forms (see Fig. 31.4). The Fab fragment is made up
of the first constant domain and the variable domain
of each of the heavy and the light chains of the anti-
body. A disulfide bond between the constant do-
mains joins the two chains. Fab fragments have
comparable affinities to the monoclonal antibodies
from which they are derived. However, their expres-
sion by bacteria may be difficult since the fragment
is too large to be expressed intact, and bacteria do
not have the machinery to form disulfide bonds cor-
rectly in the cytoplasm. Therefore, each chain must
be expressed separately and recombined.
To further reduce the size of the expressed frag-
ment, scientists removed the constant domains from
the Fab fragments, leaving the heavy and light vari-
able fragment (Fv). These fragments have higher
levels of expression in bacterial cells, due to their
smaller size, but tend not to combine because the
heavy and light chains of the Fv are not attached by
a disulfide bond and must be stabilized. The heavy
and light chains are joined by creating a short syn-
thetic peptide linker, resulting in the formation of a
single-chain variable fragment (scFv) (Fig. 31.4).
ScFvs have a number of advantages over other con-
structs. They are well expressed in bacterial systems
and can be expressed as a single gene construct.
This eliminates the need for reassociation of the
heavy and light chains. They have affinities that are
generally comparable to the original antibody, but
are somewhat lowered because they contain a single
binding domain rather than two. These scFv anti-
bodies have been used to detect pathogens such as
Streptococcus suis(De Greeff et al. 2000), Brucella
melitensis(Hayhurst et al. 2003), Bacillus(Turn-
bough Jr. 2003, Zhou et al. 2002), S. aureus(Bjer-
ketorp et al. 2002), and L. monocytogenes(Churc-
hill, unpublished data).
More recently, a different type of recombinant
fragment has been constructed, called the VHH (the
designation VHH distinguishes this heavy-chain
variable region from the corresponding VH domain
of conventional antibodies). Llamas and camels,
members of the camelid family, have antibodies that
lack a light chain but are stable under biological
conditions and have good binding affinities for anti-
gens (Churchill et al. 2002). These antibodies consist
of only a heavy-chain dimer (Fig. 31.4) (Hamers-
Casterman et al. 1993, Woolven et al. 1999). Within
the variable (VHH) domain are several amino acid
substitutions in the framework regions. These sub-
stitutions stabilize the antibody fragment, increase
its binding affinity to antigens, and increase its ex-
pression levels, while simultaneously inhibiting any
possible interactions with a VL domain (Vu et al.
1997). This allows the easy expression of VHHs as
recombinant fragments from bacterial systems.
VHHs are smaller than scFvs, and thus have higher
expression in bacterial systems. They do not contain
a linker that sometimes causes aggregation in scFv
constructs, and they are stable, remaining active in
some cases for more than a year at 4°C (unpublished
observations).Enzyme-Linked Immunosorbent Assay
(ELISA)Enzyme-linked immunosorbent assay (ELISA) is
the most common format used for immunodetection
of pathogens. By this method, most pathogens have
a detection limit of between 10^3 and 10^5 cfu/mL (de
Boer and Beumer 1999). To achieve this detection
limit often requires enrichment of the pathogens for
at least 16–24 hours before the concentration of the
pathogen is adequate for detection by ELISA (de
Boer and Beumer 1999).
There are two main ELISA formats used to detect
pathogens. In direct ELISA (Fig. 31.5A), the test
sample is coated onto a well in a microtiter plate.