404
A. Roda . P. Pasini . M. Guardigli
in environmental analytical processes beginning in the 1980s, when they were recognized as useful screening techniques for the detection of compounds of environmental regulatory concern in many countries (Van Emon 1987; Van Emon and Gerlach 1995).
The U.S. Food and Drug Administration (FDA) joined the U.S. Environmental Protection Agency (EPA) in researching immunochemical detection methods for a variety
of regulatory and monitoring applications. The high specificity of antibodies used as
biospecific recognition elements in immunochemical methods allows for the reduction in the number of pre-analytical steps, and little or no nonaqueous solvents are
used. When complex matrix samples have to be analyzed, a separation step of the
analyte from the matrix is still required before performing the quantitative step. On
the other hand, on samples with a relatively simple and stable matrix with low component variability, such as natural waters, the factors affecting the quantitative procedure can be minimized and kept under control, so that immunoassay can be carried
out directly on the sample without any dean-up procedure. Moreover, immunological
methods can be easily developed or converted in high throughput screening (HTS)
formats (for example, in 96- or 384-well microtitre plates), and optimized to provide
quick results in field applications.
17.2.1
Luminescent Immunoassays
The most common immunoassay format in environmental analysis is the ELISA (Enzyme-Linked ImmunoSorbent Assay). Enzyme immunoassays, in which a suitable
enzyme is used as a label, allow signal amplification due to the enzyme-catalysed reaction, thus obtaining a high sensitivity. Quantitation by ELISA has become more accepted in the past decade and is now a laboratory standard for environmental analysis, frequently surpassing traditional chromatographic methods in sensitivity, selectivity and cost (Van Emon and Lopez-Avila 1992). Due to the low molecular weight of
most of the organic pollutants, competitive immunoassays are the preferred format
in environmental analysis. In a typical competitive indirect heterogeneous ELISA, the
analyte contained in the sample and an analyte derivative immobilized on a solid support (e.g. a well of a 96-wells microtitre plate) compete for an enzyme-labelled antibody. After the immunoreaction and the washing steps, the enzyme-labelled antibody
bound to the solid support is quantified by means of the enzyme-catalysed reaction;
the amount of analyte contained in the sample is inversely proportional to the fraction of bound enzyme-labelled antibody. In an alternative format (direct heterogeneous ELISA), the antibody is immobilized on the solid phase, and an enzyme-labelled
analyte derivative is used.
Most of the environmental applications involve the determination of a compound
or group of compounds present in trace concentrations. Immunoassays commonly
used in environmental analysis rely on colourimetric detection procedures,
i.e. quantitation is performed through the use of a chromogenic enzyme substrate. The
intrinsic detectability of immunoassays can be further improved by the use of luminescent labels such as enzymes detectable by bio- or chemiluminescent (CL) substrates
or lanthanide chelates detectable by time-resolved fluorescence (TRF) techniques.
Chemiluminescent detection is probably the most sensitive detection principle for
enzyme labels, and the analytical sensitivity of chemiluminescent immunoassays is
A. Roda . P. Pasini . M. Guardigli
in environmental analytical processes beginning in the 1980s, when they were recognized as useful screening techniques for the detection of compounds of environmental regulatory concern in many countries (Van Emon 1987; Van Emon and Gerlach 1995).
The U.S. Food and Drug Administration (FDA) joined the U.S. Environmental Protection Agency (EPA) in researching immunochemical detection methods for a variety
of regulatory and monitoring applications. The high specificity of antibodies used as
biospecific recognition elements in immunochemical methods allows for the reduction in the number of pre-analytical steps, and little or no nonaqueous solvents are
used. When complex matrix samples have to be analyzed, a separation step of the
analyte from the matrix is still required before performing the quantitative step. On
the other hand, on samples with a relatively simple and stable matrix with low component variability, such as natural waters, the factors affecting the quantitative procedure can be minimized and kept under control, so that immunoassay can be carried
out directly on the sample without any dean-up procedure. Moreover, immunological
methods can be easily developed or converted in high throughput screening (HTS)
formats (for example, in 96- or 384-well microtitre plates), and optimized to provide
quick results in field applications.
17.2.1
Luminescent Immunoassays
The most common immunoassay format in environmental analysis is the ELISA (Enzyme-Linked ImmunoSorbent Assay). Enzyme immunoassays, in which a suitable
enzyme is used as a label, allow signal amplification due to the enzyme-catalysed reaction, thus obtaining a high sensitivity. Quantitation by ELISA has become more accepted in the past decade and is now a laboratory standard for environmental analysis, frequently surpassing traditional chromatographic methods in sensitivity, selectivity and cost (Van Emon and Lopez-Avila 1992). Due to the low molecular weight of
most of the organic pollutants, competitive immunoassays are the preferred format
in environmental analysis. In a typical competitive indirect heterogeneous ELISA, the
analyte contained in the sample and an analyte derivative immobilized on a solid support (e.g. a well of a 96-wells microtitre plate) compete for an enzyme-labelled antibody. After the immunoreaction and the washing steps, the enzyme-labelled antibody
bound to the solid support is quantified by means of the enzyme-catalysed reaction;
the amount of analyte contained in the sample is inversely proportional to the fraction of bound enzyme-labelled antibody. In an alternative format (direct heterogeneous ELISA), the antibody is immobilized on the solid phase, and an enzyme-labelled
analyte derivative is used.
Most of the environmental applications involve the determination of a compound
or group of compounds present in trace concentrations. Immunoassays commonly
used in environmental analysis rely on colourimetric detection procedures,
i.e. quantitation is performed through the use of a chromogenic enzyme substrate. The
intrinsic detectability of immunoassays can be further improved by the use of luminescent labels such as enzymes detectable by bio- or chemiluminescent (CL) substrates
or lanthanide chelates detectable by time-resolved fluorescence (TRF) techniques.
Chemiluminescent detection is probably the most sensitive detection principle for
enzyme labels, and the analytical sensitivity of chemiluminescent immunoassays is