antibody with specificity towards particular antigen. The subsequent reaction
produces a detectable signal, most commonly a color change in the substrate. The
sample with an unknown amount of antigen is immobilized on a solid support
known as a polystyrene microtiter plate either nonspecifically or specifically. After
the antigen is immobilized, the primary antibody (detection antibodies) is added,
forming a complex with the antigen. The primary antibody can be covalently linked
to an enzyme or can itself be detected by a secondary antibody that is linked to an
enzyme through bioconjugation. The main enzymes used are horseradish peroxidase, alkaline phosphatase, and β-galactosidase. Between each step, the plate is
typically washed with a mild detergent solution to remove any proteins or antibodies
that are nonspecifically bound. After the final wash step, the plate is developed by
adding an enzymatic substrate to produce a visible signal, which indicates the
quantity of antigen in the sample.
9.2.2.1 Basic Steps for Developing and Running an Immunoassay (Karen
et al. 2012)
1. Establish assay critical success factors (i.e., sensitivity required).
2. Ensure that appropriate antibody and antigen reagents are available.
3. Adsorb antigen or capture antibody to a solid surface.
4. Wash off unbound reagents.
5. Block nonspecific binding sites to reduce background.
6. Incubate the secondary antibody with the sample.
7. Wash off unbound reagents.
8. Incubate secondary antibody conjugate with sample.
9. Wash off unbound reagents.
10. Incubate substrate to generate signal.
11. Calibrate curve fitting, data analysis, and quantitation by nonlinear regression.
Enzyme-linked immunosorbent assay (ELISA) is a widely used immunoassay for
environmental purposes. Three different types of ELISA are carried out according to
different formats: direct competitive, indirect competitive, or sandwich type. Competitive assays are most common and can be performed in different ways. Analyte
and the tracer (direct competitive ELISA) or analyte and the immobilized ligand
(indirect ELISA) may compete for a limited number of binding sites. Sandwich-type
ELISA is a noncompetitive assay, in which the analyte is recognized by two different
antibodies, immobilized Ab and marker Ab (Harris 1999; Farre et al. 2005). Flowinjection immunoassay (FIIA) is a technique, based on the introduction of the sample
into carrier stream, which enters the reaction chamber where the immunoreaction
takes place. FIIA has been successfully used for detection of different pollutants,
e.g., triazines. At present, this method is integrated into different immunosensors.
Immunoassay and other immune techniques are the powerful and elegant techniques
for rapid detection of environmental pathogen and its toxins. They also provide
accurate and convenient means of detection of adulteration and authentic assay.
These assays are fast and relatively inexpensive. Immunoassays are not as susceptible to matrix effects as PCR assays. Meng and Doyle (2002) and Taitt et al. (2004)
9 Development of Modern Tools for Environmental Monitoring of Pathogens and. . .
195
produces a detectable signal, most commonly a color change in the substrate. The
sample with an unknown amount of antigen is immobilized on a solid support
known as a polystyrene microtiter plate either nonspecifically or specifically. After
the antigen is immobilized, the primary antibody (detection antibodies) is added,
forming a complex with the antigen. The primary antibody can be covalently linked
to an enzyme or can itself be detected by a secondary antibody that is linked to an
enzyme through bioconjugation. The main enzymes used are horseradish peroxidase, alkaline phosphatase, and β-galactosidase. Between each step, the plate is
typically washed with a mild detergent solution to remove any proteins or antibodies
that are nonspecifically bound. After the final wash step, the plate is developed by
adding an enzymatic substrate to produce a visible signal, which indicates the
quantity of antigen in the sample.
9.2.2.1 Basic Steps for Developing and Running an Immunoassay (Karen
et al. 2012)
1. Establish assay critical success factors (i.e., sensitivity required).
2. Ensure that appropriate antibody and antigen reagents are available.
3. Adsorb antigen or capture antibody to a solid surface.
4. Wash off unbound reagents.
5. Block nonspecific binding sites to reduce background.
6. Incubate the secondary antibody with the sample.
7. Wash off unbound reagents.
8. Incubate secondary antibody conjugate with sample.
9. Wash off unbound reagents.
10. Incubate substrate to generate signal.
11. Calibrate curve fitting, data analysis, and quantitation by nonlinear regression.
Enzyme-linked immunosorbent assay (ELISA) is a widely used immunoassay for
environmental purposes. Three different types of ELISA are carried out according to
different formats: direct competitive, indirect competitive, or sandwich type. Competitive assays are most common and can be performed in different ways. Analyte
and the tracer (direct competitive ELISA) or analyte and the immobilized ligand
(indirect ELISA) may compete for a limited number of binding sites. Sandwich-type
ELISA is a noncompetitive assay, in which the analyte is recognized by two different
antibodies, immobilized Ab and marker Ab (Harris 1999; Farre et al. 2005). Flowinjection immunoassay (FIIA) is a technique, based on the introduction of the sample
into carrier stream, which enters the reaction chamber where the immunoreaction
takes place. FIIA has been successfully used for detection of different pollutants,
e.g., triazines. At present, this method is integrated into different immunosensors.
Immunoassay and other immune techniques are the powerful and elegant techniques
for rapid detection of environmental pathogen and its toxins. They also provide
accurate and convenient means of detection of adulteration and authentic assay.
These assays are fast and relatively inexpensive. Immunoassays are not as susceptible to matrix effects as PCR assays. Meng and Doyle (2002) and Taitt et al. (2004)
9 Development of Modern Tools for Environmental Monitoring of Pathogens and. . .
195