28 In Situ Detection of Viral Nucleic Acids by Chemiluminescence
347
the detection of a few zeptomoles of AP using a photomultiplier tube
(Bronstein et al. 1990) or attomoles of enzyme using a video camera
(Roda et al. 1996) and have a glowing kinetics with a steady-state light
emission lasting several minutes, which permits easy handling and analysis of the samples with an increased detection sensitivity in the analysis
of nucleic acids.
For horseradish peroxidase (HRP) chemiluminescent detection, luminol-based reagents are also commercially available [e.g. the enhanced chemiluminescent reagent ECL (luminolJenhancer/HzO z ) (Amersham, Amersham, UK) and SuperSignal Ultra (luminolJenhancerlstable peroxide)
(Pierce, Rockford, IL, USA)]. Both reagents contain substances, such as
p-iodophenol, which enhance the light production deriving from the
HRP-catalyzed oxidation of luminol by hydrogen peroxide, thus permitting detection of HRP with very high efficiency. The kinetics is of a glowtype with a steady-state light emission maintained for at least 10 min (Matthews et al. 1985, Thorpe and Kricka 1986, Thorpe and Kricka, 1987).
Continuing improvements in chemiluminescent substrates have recently been matched by new developments in photon imaging instrumentation such as high performance luminographs based on a CCD video
camera or high dynamic range pick up tube (Saticon) combined with a
video amplifier. These instruments not only allow a quantification of
emitted light at a single photon level, but also permit localization of
the chemiluminescent emission on a target surface (Scott and Inaba
1989, Wick 1989, Hooper and Ansorge 1991, Brauer et al. 1993, Roda
et al. 1996). Moreover, connecting the luminograph to an optical microscope, it is possible to localize the light emission on a target surface or
inside tissues or cells (Hiraoka et al. 1987, Mueller-Klieser et al. 1988, Hawkins and Cumming 1990, Lorimier et al. 1993, Mueller-Klieser and Walenta 1993, Musiani et al. 1996a, Roda et al. 1998, Pasini et al. 1998). The evolution of photon counting imaging together with the continuous development of chemiluminescent substrates for AP and HRP has recently led to
the development of chemiluminescence in situ hybridization assays for
the detection of viral genomes having AP and HRP as reporter molecules
(Musiani et al. 1998).
The chemiluminescent in situ hybridization assay for viral genome detection has the advantage of combining the specificity of labeled probes,
the sensitivity of enzymatic chemiluminescent substrates and the spatial
morphological resolution and localization of the signal of the in situ hybridization.
347
the detection of a few zeptomoles of AP using a photomultiplier tube
(Bronstein et al. 1990) or attomoles of enzyme using a video camera
(Roda et al. 1996) and have a glowing kinetics with a steady-state light
emission lasting several minutes, which permits easy handling and analysis of the samples with an increased detection sensitivity in the analysis
of nucleic acids.
For horseradish peroxidase (HRP) chemiluminescent detection, luminol-based reagents are also commercially available [e.g. the enhanced chemiluminescent reagent ECL (luminolJenhancer/HzO z ) (Amersham, Amersham, UK) and SuperSignal Ultra (luminolJenhancerlstable peroxide)
(Pierce, Rockford, IL, USA)]. Both reagents contain substances, such as
p-iodophenol, which enhance the light production deriving from the
HRP-catalyzed oxidation of luminol by hydrogen peroxide, thus permitting detection of HRP with very high efficiency. The kinetics is of a glowtype with a steady-state light emission maintained for at least 10 min (Matthews et al. 1985, Thorpe and Kricka 1986, Thorpe and Kricka, 1987).
Continuing improvements in chemiluminescent substrates have recently been matched by new developments in photon imaging instrumentation such as high performance luminographs based on a CCD video
camera or high dynamic range pick up tube (Saticon) combined with a
video amplifier. These instruments not only allow a quantification of
emitted light at a single photon level, but also permit localization of
the chemiluminescent emission on a target surface (Scott and Inaba
1989, Wick 1989, Hooper and Ansorge 1991, Brauer et al. 1993, Roda
et al. 1996). Moreover, connecting the luminograph to an optical microscope, it is possible to localize the light emission on a target surface or
inside tissues or cells (Hiraoka et al. 1987, Mueller-Klieser et al. 1988, Hawkins and Cumming 1990, Lorimier et al. 1993, Mueller-Klieser and Walenta 1993, Musiani et al. 1996a, Roda et al. 1998, Pasini et al. 1998). The evolution of photon counting imaging together with the continuous development of chemiluminescent substrates for AP and HRP has recently led to
the development of chemiluminescence in situ hybridization assays for
the detection of viral genomes having AP and HRP as reporter molecules
(Musiani et al. 1998).
The chemiluminescent in situ hybridization assay for viral genome detection has the advantage of combining the specificity of labeled probes,
the sensitivity of enzymatic chemiluminescent substrates and the spatial
morphological resolution and localization of the signal of the in situ hybridization.