Diagnostic tests
Enzyme-Linked Immunoassay Tests
Conventional methods for recovering sal-
monella require 3 to 4 days to yield negative
results and up to 7 days for a positive result.
Furthermore, a high level of technical skill is
required to perform these tests. Because of the
time and skill required, several rapid methods
for detecting Salmonellahave been developed
such as enzyme-linked immunosorbent assays
(ELISAs), immunodiffusion methods, immu-
nomagnetic bead ELISAs, nucleic acid
hybridization methods, and polymerase
chain reaction methods (Shearer et al., 2001).
Furthermore, automated immunodiagnostic
assays have been developed. Yeh et al. (2002)
concluded that the VIDAS-SLM automated
method is a rapid screening technique and a
potential alternative to the time- and labor-
intensive culture method. Goodridge et al.
(2003) developed a rapid MPN-ELISA for the
detection and enumeration of Salmonella
typhimuriumin poultry processing wastewater.
A common assay for detecting specific
pathogens and/or their toxins is immunolog-
ical (antigen-antibody) reactions. Antigens
are the specific constituents of a cell or toxin
that induce an immune response and interact
with a specific antibody; whereas, antibodies
are immunoglobulins that bind specifically
to antigens. Either monoclonal or polyclonal
antibodies are used in immunobased assays.
Monoclonals are a single type of antibody
with a high affinity for a specific target anti-
gen epitope. A polyclonal antibody is a set of
different antibodies specific for an antigen
but able to recognize different epitopes of
the antigen. The advantages of these assays
are rapid results, increased sensitivity and
specificity, and decreased costs (Phebus and
Fung, 1994).
Enzyme-linked immunoassays (ELISAs)
have been effective in detecting pathogens
and are easy to conduct. These systems are
formatted to consist of antibodies attached
to a solid support, such as the walls of a
microtiter plate or a plastic dipstick. An
enrichment culture is added to the solid sup-
port while antibodies bind target antigens in
the sample. A sandwich format is used fre-
quently, in which a second enzyme-labeled
antibody is added to the sample, followed by
a reactive substrate, to produce a positive
color reaction. If the target antigens are not
present, the labeled antibody will not attach
and no color reaction occurs.
An efficient and sensitive method of ana-
lyzing samples for pathogens is immunoblot-
ting. The common procedure involves an
enrichment culture that is spotted onto a
solid support (i.e., nitrocellulose paper), with
the remaining protein binding areas of the
paper blocked by dipping in a protein
solution such as bovine serum albumin or
reconstituted dry milk. An enzyme-labeled
antibody solution specific for the target
pathogen is applied, and a substrate for the
enzyme is added after washing to remove the
unbound antibody. If the labeled antibody is
present, due to attachment to the target anti-
gen, a color reaction will indicate a positive
sample. This procedure can be modified for
use in conjunction with other methods, such
as the HGMF system.
Another technique for pathogen detection
is the use of superparamagnetic microspheres
coated with an antibody specific to a target
antigen. The sample is selectively enriched,
and a small amount (approximately 10 mL) of
the enrichment culture is transferred to a test
tube. The antibody-coated beads are added
and shaken gently for a short period. Then a
magnetic particle concentrator is used to sep-
arate beads from the sample homogenate.
After reconstitution in a buffer, the beads are
spread-plated onto a selective agar to observe
growth of the target pathogen. If present in
the original sample, presumptive colonies