Food Biochemistry and Food Processing

31 Emerging Bacterial Foodborne Pathogens and Methods of Detection 735

plate, allowing a more intense signal. This variation
is known as a sandwich ELISA (Hess et al. 1998).
More commonly, a competitive ELISA (Fig.
31.5B) is used to detect the presence of pathogens,
since this method is less susceptible to nonspecific
background binding. In this format, a known amount
of antigen is coated onto the plate. The sample to be
tested is incubated with the primary antibody and
then added to the blocked well. After washing, the
secondary antibody is added to the well, and detec-
tion is performed as mentioned above. In this case,
the more free antigen present in the test solution, the
less signal will be detected after treatment with the
secondary antibody since the majority of the pri-
mary antibody will have bound to the soluble anti-
gen, and is consequently washed away. The amount
of inhibition can be compared to an inhibition curve
created with known amounts of antigen in the sam-
ples, thus allowing a quantitative determination of
the amount of pathogen present in the test sample
via interpolation using the standard curve (Hess et
al. 1998).

Fluorescently Labeled Immunoassays

The ELISA assay can be made more sensitive using
fluorescent labels on the antibodies. This type of
labeling also decreases the time required for detec-
tion by eliminating the need for a colorimetric reac-
tion as the final step of the ELISA. The labeling
does, however, increase the cost of the assay.
Going back to the example of detecting L. mono-
cytogenes, Sewell et al. (Sewell et al. 2003) used a
modified version of an ELISA system with a fluores-
cent reporter dye. After 52 hours of culture en-
richment, the authors carried out the ELFA (enzyme-
linked fluorescent assay) procedure. The accuracy
of the positive results was 97%, with positive sam-
ples requiring confirmation by plating on Listeria-
selective media (Sewell et al. 2003). This final
step, unfortunately, also adds more time to the pro-
cedure.
Similar to this approach, Dunbar et al. (2003)
used microspheres with different spectral labels,
coated with antibodies to each of E. coliO157:H7,
Salmonella typhimurium, C. jejuni, and L. monocy-
togenes,respectively, to detect the pathogens after
enrichment and amplification. The spheres were
sorted by their spectral labels using a fluorescent
bead sorter (Luminex Labmap system, Austin,
Texas), and the pathogens were detected using a sec-

ondary antibody labeled with a fluorophore. The

assay is able to detect between 2.5 and 500 organ-

isms/mL depending on the species (Dunbar et al.

2003). This system, although expensive, allows mul-

tiple pathogens to be detected simultaneously.

Immunomagnetic Assays

Immunomagnetic assays make use of antibody

specificity for a pathogen to concentrate the path-

ogen before other methods are used to amplify and

identify the bacteria. Hudson et al. (2001) used the

procedure to isolate L. monocytogenesdirectly from

ham. In this procedure, the food was homogenized

with some growth medium, the particulate matter

removed, and after a number of washes, particles of

bacterial size were pelletted and resuspended in a

low volume. Commercial immunomagnetic beads

coated with an anti-Listeriaantibody were added to

the solution and incubated to allow binding of the L.

monocytogenesto the beads. The beads were trap-

ped on a magnet, washed, and the DNA extracted

for amplification ofL. monocytogenes–specific genes

by PCR. The immunomagnetic separation and con-

centration cut the detection time to about 1 day, at

least for ham, but it is limited in terms of sensitivity,

since the recovery of cells on the beads was only

about 20% of those initially added (Hudson et al.

2001). Immunomagnetic separation on average al-

lowed detection of 1–2 cfu/g food sample, but the

results were somewhat variable in terms of sensitiv-

ity (detection limit from 0.1 cfu/g to greater than 5.7

cfu/g) (Hudson et al. 2001), making this method

promising, but not ready to be used by the food

industry until the efficiency of immunomagnetic iso-

lation is improved.

Surface Plasmon Resonance (SPR)

Surface plasmon resonance (SPR) relies on an opti-

cal phenomenon to measure the binding kinetics

between molecules (Fig. 31.6A). SPR can detect

interactions between unmodified proteins and meas-

ure the kinetics in real time (Hashimoto 2000). The

most popular equipment for this method is the

BIAcore machine (biomolecular interaction assay),

in which one of the proteins is coated on a dispos-

able chip that is placed in the machine. Sample

buffer is passed over the chip, and its flow is inter-

rupted by the passage of discreet amounts of sample.

The sensing mechanism of the machine measures