730 Part VII: Food Safety
structural dynamics of microbial communities.
FISH can be used in phylogenetic studies and in
assessing the spatial distribution of target microbes
in communities such as biofilms (Wagner et al.
1998). The method has only recently been applied to
detecting and analyzing microbes in food. To per-
form FISH, a sample is fixed to a slide, membrane
filter, or well using ethanol. In the case of Gram-
positive organisms such as Listeria, the fixed cells
are permeabilized with proteinase K so that fluores-
cent probes are able to enter the cell. Specific probes
are created that are complementary to DNA or
rRNA inside the cell, and labeled with a fluorescent
tag such as Cy3, Cy5, or CFLUOS. The fixed cells
are incubated with the labeled probes under condi-
tions that allow labeling of the microbes of interest
but not the background microflora. The labeled cells
can then be visualized with a fluorescence micro-
scope (Ootsubo et al. 2003, Schmid et al. 2003,
Wagner et al. 1998). For working with Listeriain
milk samples, Wagner et al. (1998) enriched the bac-
teria in Listeriaenrichment broth for 1, 2, or 7 days
before analysis. Using primers specific for the 16S
rRNA genes, the group was able to detect Listeria
reliably on the genus level, allowing for further
analysis by competitive PCR.
IMMUNOASSAY-BASEDMETHODS
Immunoassays are based on the natural affinity of
antibodies for their antigens, regardless of whether
the antibody is against a hapten, a protein, or a car-
bohydrate on the surface of a cell. These assays are
fast, and relatively inexpensive. They allow accurate
detection of contaminants after very little sample
purification (Hall et al. 1989b). Immunoassays are
not as susceptible to matrix effects as PCR assays;
samples such as river water can be analyzed directly
by ELISA (enzyme-linked immunosorbent assay;
Hall et al. 1989a). In addition, immunoassays can be
used to provide information in a real-time manner
and allow for a timely response if quantities of the
pathogen are high enough (Meng and Doyle 2002).
The biggest problems with immunoassay-based
methods are the low sensitivity of the assays, the
low affinity of the antibody to the pathogen or other
analytes being measured, and potential interference
from contaminants. Improvements in these areas
will likely expand the use of immunoassays in a
variety of fields, including the food industry. A com-
parison of various immunoassay methods used to
detect foodborne pathogens is presented in Table
31.5.Types of Antibodies Used in Detection of
PathogensA number of antibody types and formats are avail-
able for immunodetection. These include conven-
tional and heavy-chain antibodies, as well as poly-
clonal, monoclonal, or recombinant antibodies (see
Fig. 31.4).
Polyclonal antibodies have been used as detection
vehicles for several decades (Breitling and Dübel
1999). These antibodies are raised by immunizing
an animal host with the antigen several times, then
harvesting serum from the animal. The serum ob-
tained contains a mixture of antibodies, most of
which do not bind to the antigen and were present
before immunization. However, if the antigen is im-
munogenic, containing a number of epitopes to
which antibodies can bind, this is a simple way of
obtaining a detection reagent for performing im-
munoassays.
In 1975, Köhler and Milstein were the first to iso-
late monoclonal antibodies from hybridoma cells
(Köhler and Milstein 1975). Monoclonal antibodies
are more useful for the specific detection of a mole-
cule than polyclonal antibodies. Raising a mono-
clonal antibody also begins with the immunization
of an animal. Once the immune response has been
maximized, the B-cells producing the antibodies are
isolated from the spleen of the immunized animal
and fused with myeloma cancer cells, to produce a
hybridoma cell that has the immortality of a cancer
cell and the antibody producing capability of the B-
cell.
Monoclonal antibodies have a number of advan-
tages over polyclonal antibodies. First, hybridoma
cell lines expressing monoclonal antibodies can be
cultured indefinitely in vitro to provide a continuous
supply of homogeneous, well-characterized antibod-
ies. Clones can be selected with different specifici-
ties and affinities for a molecule, or a family of mo-
lecules. Nonspecific antibodies are removed during
the selection process, thereby limiting interference in
the assay (Deschamps and Hall 1990, Deschamps et
al. 1990). However, monoclonal antibodies are ex-
pensive to produce, requiring a skillful technician
and specialized growth apparatus for tissue culturing,