46
S. Iftekhar et al.
(Zhang et al. 2006). This method was based on an immunoextraction column that
utilized monoclonal antibodies against diuron that were contained in a sol-gel support. This immunoextraction support was coupled online with a reversed-phase silica
monolith analytical column. This method gave a detection limit of 0.9 ng L
−1 for
diuron and a recovery for this analyte of 93% (Zhang et al. 2006). Immunoextraction can also be coupled online with GC (Hage 1998). This approach has been used
for the quantification of atrazine in wastewater (Dalluge et al. 1999). Monoclonal
antibodies against atrazine were immobilized onto beaded cellulose for this application. After the sample had been injected onto the antibody-containing support, the
retained analytes were desorbed and passed onto a cartridge that contained a nonpolar styrene-divinylbenzene copolymer for collection of the eluting compounds.
The retained compounds were later desorbed from the copolymer cartridge by using
ethyl acetate and introduced onto a GC system for separation and analysis. The elution of atrazine and retained compounds was monitored by using a flame ionization
detector and nitrogen phosphorous detector, which gave mass detection limits for
atrazine of 120–170 pg and 15 pg, respectively (Dalluge et al. 1999).
2.2.4 Chromatographic Immunoassays
Antibodies have also been used in chromatographic systems for the binding and
indirect detection of emerging contaminants in the environment. This is accomplished
by using a technique which is known as a chromatographic immunoassay (Hage 1998;
Nelson and Hage 2006). This type of technique is advantageous for the detection
of trace analytes that are not present in sufficient quantities to be detected directly
(Hage and Nelson 2001; Nelson and Hage 2006).
In a competitive binding immunoassay, the analyte and a fixed amount of a labeled
analog of the analyte are allowed to compete for a limiting number of antibodies.
The amount of labeled analog that is bound to the antibodies, or that remains free
in solution, is then measured and used to determine the amount of analyte that was
present in the sample (Hage 1998). There are a variety of formats that have been
reported for conducting this type of method by chromatography (Hage et al. 1993,
1999; Hage and Nelson 2001; Nelson et al. 2003). One approach is to mix the antibodies with the sample and labeled analog and then capture the antibodies and their
complexes with the analog or analyte by a column, thus separating the free and
bound fractions of the labeled analog, as illustrated in Fig. 2.5. This approach has
been used in a method for the analysis of β-lactam antibiotics in the effluents of
sewage treatment plants (Benito-Pena et al. 2005). This approach used polyclonal
antibodies prepared against 6-aminopencillanic acid (i.e., a common structural component of β-lactam antibiotics); a labeled analog that contained a fluorescent tag;
and a column-containing protein A/G (i.e., an antibody-binding agent) to capture
the antibodies and their immunocomplexes. Upon desorption of the antibody-bound
labeled analog and analyte from the protein A/G column, the fluorescence generated
S. Iftekhar et al.
(Zhang et al. 2006). This method was based on an immunoextraction column that
utilized monoclonal antibodies against diuron that were contained in a sol-gel support. This immunoextraction support was coupled online with a reversed-phase silica
monolith analytical column. This method gave a detection limit of 0.9 ng L
−1 for
diuron and a recovery for this analyte of 93% (Zhang et al. 2006). Immunoextraction can also be coupled online with GC (Hage 1998). This approach has been used
for the quantification of atrazine in wastewater (Dalluge et al. 1999). Monoclonal
antibodies against atrazine were immobilized onto beaded cellulose for this application. After the sample had been injected onto the antibody-containing support, the
retained analytes were desorbed and passed onto a cartridge that contained a nonpolar styrene-divinylbenzene copolymer for collection of the eluting compounds.
The retained compounds were later desorbed from the copolymer cartridge by using
ethyl acetate and introduced onto a GC system for separation and analysis. The elution of atrazine and retained compounds was monitored by using a flame ionization
detector and nitrogen phosphorous detector, which gave mass detection limits for
atrazine of 120–170 pg and 15 pg, respectively (Dalluge et al. 1999).
2.2.4 Chromatographic Immunoassays
Antibodies have also been used in chromatographic systems for the binding and
indirect detection of emerging contaminants in the environment. This is accomplished
by using a technique which is known as a chromatographic immunoassay (Hage 1998;
Nelson and Hage 2006). This type of technique is advantageous for the detection
of trace analytes that are not present in sufficient quantities to be detected directly
(Hage and Nelson 2001; Nelson and Hage 2006).
In a competitive binding immunoassay, the analyte and a fixed amount of a labeled
analog of the analyte are allowed to compete for a limiting number of antibodies.
The amount of labeled analog that is bound to the antibodies, or that remains free
in solution, is then measured and used to determine the amount of analyte that was
present in the sample (Hage 1998). There are a variety of formats that have been
reported for conducting this type of method by chromatography (Hage et al. 1993,
1999; Hage and Nelson 2001; Nelson et al. 2003). One approach is to mix the antibodies with the sample and labeled analog and then capture the antibodies and their
complexes with the analog or analyte by a column, thus separating the free and
bound fractions of the labeled analog, as illustrated in Fig. 2.5. This approach has
been used in a method for the analysis of β-lactam antibiotics in the effluents of
sewage treatment plants (Benito-Pena et al. 2005). This approach used polyclonal
antibodies prepared against 6-aminopencillanic acid (i.e., a common structural component of β-lactam antibiotics); a labeled analog that contained a fluorescent tag;
and a column-containing protein A/G (i.e., an antibody-binding agent) to capture
the antibodies and their immunocomplexes. Upon desorption of the antibody-bound
labeled analog and analyte from the protein A/G column, the fluorescence generated