544 ◾ Fundamental Food Microbiology
is provided, microbes will metabolize and produce byproducts, such as acid or ionic species, that
will reduce a colorless indicator like tetrazolium salt to a purple product, generating unique metabolic fingerprint patterns. This method requires pure cultures and certain physiological properties,
such as gram-staining, oxygen requirements, and growth temperature optima, before applying
this technique for detection. Miniature biochemical test kits (API strips) are available commercially for different microorganisms. In a more advanced metabolic fingerprinting system, a 96-well
plate containing multiple substrates, sugars, or amino acids are used, and the substrate utilization
patterns are compared with the database for specific identification of an organism in the MicroLog
system (Biolog, Inc.).
Immunoassays for Rapid Detection of Pathogens 12,13
Several rapid and automated methods have been developed that rely on the specific antigenantibody reaction and production of agglutination, color formation from a chromogenic substrate,
formation of an immunoband, or fluorescence. Pathogen diagnosis relies heavily on antibodies.
Antibody-based assay methods are simple and less cumbersome and have and easy-to-interpret
results. These methods allow detection of intact cells, their secreted toxins, or metabolic byproducts. However, this method may not be able to differentiate between live and dead cells although
live cells are of concern in food safety. Active culturing coupled with immunoassay can overcome
such a problem. In addition, microbes are routinely exposed to sublethal injury or stress conditions, such as acidic pH, osmotic stress (salt), antimicrobial preservatives, storage temperatures,
and heat-shock conditions in food, which may alter morphology and affect physiology resulting in
aberrant antigen expression and a weakened signal during antibody-based detection.
The success of the immunoassay depends on the availability of an antigen-specific antibody.
More importantly, the knowledge of antibodies’ binding affinity and avidity should be known
before employing them for a specific application. Two types of antibodies are used commonly:
polyclonal and monoclonal. Polyclonal antibodies (PAb) contain an assortment of antibody molecules having different cellular origins and made against different epitopes of the same antigen,
and therefore they may show some cross-reactions with antigens from different microbes. To overcome this problem, PAb can be made epitope-specific by carefully designing an epitope-specific
synthetic peptide. The peptide is then conjugated to a large carrier protein for immunization of
animals, such as rabbits or goats, for antibody generation. The quality of such antibodies may
vary from batch to batch, which may affect the end result. In contrast, monoclonal antibodies
(MAb) are homogeneous and are highly specific because they are produced by a single, preselected
B-lymphocyte clone that is fused with a cancerous immortal myeloma cell to form a hybridoma
line. Immunoassay experiments designed carefully to employ both polyclonal and monoclonal
antibodies in a sandwich format can provide the best signal and the highest specificity with reproducible data.
Antibodies are widely used in various assay formats, such as immunomagnetic separation
methods for capture and concentration of microbes, enzyme or fluorescence immunoassays, and
immunosensor applications, which are discussed below.
Immunomagnetic Separation (IMS) 5,11,14
Small magnetic particles coated with antibody molecules are used in the capture and concentration of target pathogens from a mixture by a process known as immunomagnetic separation (IMS)
(Figure 42.2). The magnetic particles used for these applications are superparamagnetic; that is,
is provided, microbes will metabolize and produce byproducts, such as acid or ionic species, that
will reduce a colorless indicator like tetrazolium salt to a purple product, generating unique metabolic fingerprint patterns. This method requires pure cultures and certain physiological properties,
such as gram-staining, oxygen requirements, and growth temperature optima, before applying
this technique for detection. Miniature biochemical test kits (API strips) are available commercially for different microorganisms. In a more advanced metabolic fingerprinting system, a 96-well
plate containing multiple substrates, sugars, or amino acids are used, and the substrate utilization
patterns are compared with the database for specific identification of an organism in the MicroLog
system (Biolog, Inc.).
Immunoassays for Rapid Detection of Pathogens 12,13
Several rapid and automated methods have been developed that rely on the specific antigenantibody reaction and production of agglutination, color formation from a chromogenic substrate,
formation of an immunoband, or fluorescence. Pathogen diagnosis relies heavily on antibodies.
Antibody-based assay methods are simple and less cumbersome and have and easy-to-interpret
results. These methods allow detection of intact cells, their secreted toxins, or metabolic byproducts. However, this method may not be able to differentiate between live and dead cells although
live cells are of concern in food safety. Active culturing coupled with immunoassay can overcome
such a problem. In addition, microbes are routinely exposed to sublethal injury or stress conditions, such as acidic pH, osmotic stress (salt), antimicrobial preservatives, storage temperatures,
and heat-shock conditions in food, which may alter morphology and affect physiology resulting in
aberrant antigen expression and a weakened signal during antibody-based detection.
The success of the immunoassay depends on the availability of an antigen-specific antibody.
More importantly, the knowledge of antibodies’ binding affinity and avidity should be known
before employing them for a specific application. Two types of antibodies are used commonly:
polyclonal and monoclonal. Polyclonal antibodies (PAb) contain an assortment of antibody molecules having different cellular origins and made against different epitopes of the same antigen,
and therefore they may show some cross-reactions with antigens from different microbes. To overcome this problem, PAb can be made epitope-specific by carefully designing an epitope-specific
synthetic peptide. The peptide is then conjugated to a large carrier protein for immunization of
animals, such as rabbits or goats, for antibody generation. The quality of such antibodies may
vary from batch to batch, which may affect the end result. In contrast, monoclonal antibodies
(MAb) are homogeneous and are highly specific because they are produced by a single, preselected
B-lymphocyte clone that is fused with a cancerous immortal myeloma cell to form a hybridoma
line. Immunoassay experiments designed carefully to employ both polyclonal and monoclonal
antibodies in a sandwich format can provide the best signal and the highest specificity with reproducible data.
Antibodies are widely used in various assay formats, such as immunomagnetic separation
methods for capture and concentration of microbes, enzyme or fluorescence immunoassays, and
immunosensor applications, which are discussed below.
Immunomagnetic Separation (IMS) 5,11,14
Small magnetic particles coated with antibody molecules are used in the capture and concentration of target pathogens from a mixture by a process known as immunomagnetic separation (IMS)
(Figure 42.2). The magnetic particles used for these applications are superparamagnetic; that is,