Biology Reference
In-Depth Information
Figure 8.3
A typical assay designed to detect an antigen in a complex protein mixture,
the antigen is immobilized either by direct adsorption or via an antibody adsorbed to
the wells of a microplate. The plate is blocked and the antigen is probed with a spe-
cific detection antibody. The detection antibody may be directly labeled with a signal-
generating enzyme or fluorophore or it may be secondarily probed with an enzyme- or
fluor-labeled secondary antibody. For enzymatic detection, the appropriate enzyme
substrate is added. The signal observed is proportional to the amount of antigen in
the sample. Source:
Image taken from
http://www.piercenet.com/iles/TR0065-ELISA-
Combining immunological detection with selective preenrichment can
increase the recovery and detection of
E. coli
O157
31,32
and is necessary to
obtain sensitive detection of contaminant organisms from samples.
33,34
This
aspect of sample processing, along with other sample concentration and
isolation methods, was discussed in Chapter 4.
Many immunology-based methods can be coupled with other meth-
ods for pathogen detection, such as immunomagnetic separation (IMS)
on magnetic beads (
Fig. 8.4
), has been coupled with MALDI-TOF spec-
trometry for detection of
Staphylococcal
enterotoxin B and with flow
cytometry for detection of
Listeria monocytogenes
.
36
Setterington et al.
37
isolated
E. coli
O157:H7 cells via IMS and labeled them with biofunc-
tionalized electroactive polyaniline (immuno-PANI). Subsequently, the
labeled cell complexes were deposited onto a carbon electrode sensor
and pulled to the electrode surface by an external magnetic field to
amplify the electrochemical signal generated by the polyaniline. The
developed biosensor could detect as low as 7 cfu of
E. coli
O157:H7 in
70 min (from sampling to detection).
8.2.3. Nucleic acids
Nucleic acids (DNA and RNA) are polymers composed of nucleotides.
DNA's role as the hereditary material is supported by its structure, which
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