356
MONICA MUSIANI et al.
mM NaCl, 50 mM MgCI 2 , pH 9.5) and then incubate for 20 min with 20-40
JlI of a dioxetane phosphate-based reagents (e.g. CSPD, COP-star, LumiPhos Plus) prepared according to the manufacturer's instructions and acquire the light emission after having removed the chemiluminescent substrate.
In the double chemiluminescence reaction, the enzymatic reaction for
HRP is usually performed before that of AP. This is done because the HRPI
luminol reaction rapidly reaches a steady state light output which is maintained for a short time, then the signal decreases while the AP/dioxetane
reaction slowly reaches the steady-state light emission which then lasts for
a relatively long time. Short washes to remove the HRP substrate are thus
sufficient to avoid any interfering photon emission during the AP reaction. Even if, for reasons of rapidity of the assay, the enzymatic reaction
for HRP is usually performed before that of AP, consistent results can also
be obtained even when the substrate reaction sequence is reversed.
Results
The advantages of chemiluminescence in situ hybridization are mainly:
improved sensitivity, quantification of the data, objectivity of the evaluation and digital imaging of the results.
Improved
sensitivity
Chemiluminescence in situ hybridization proved to be very sensitive,
being able to detect as few as 10 to 50 viral genome copies in infected cells
using both biotynilated probes and digoxigenin labeled probes with HRP
and AP chemiluminescent detections respectively. In fact, positive cherniluminescent signals were obtained in HeLa cells which are known to contain about 10 to 50 integrated genome copies of human papillomavirus
(HPV) 18 (Lorimier et al. 1996, Musiani et al. 1997). Chemiluminescence
in situ hybridization has proved more sensitive than in situ hybridization
followed by colorimetric detection and almost as sensitive as in situ hybridization followed by 35S autoradiography; in fact, the chemiluminescence positivity of the results obtained from the detection of HPV
DNA in HeLa cells was not obtained with colorimetric detection (Lorimier
et al. 1993, Musiani et al. 1997) while chemiluminescent signal resolution
was comparable to that provided by 35S autoradiography (Lorimier et al.
1996). Moreover with chemiluminescence in situ hybridization to detect
B19 parvovirus DNA in several samples of bone marrow cells, all the positive specimens from patients with a diagnosed B19 infection proved positive with a higher number of positive cells/specimen in comparison with
MONICA MUSIANI et al.
mM NaCl, 50 mM MgCI 2 , pH 9.5) and then incubate for 20 min with 20-40
JlI of a dioxetane phosphate-based reagents (e.g. CSPD, COP-star, LumiPhos Plus) prepared according to the manufacturer's instructions and acquire the light emission after having removed the chemiluminescent substrate.
In the double chemiluminescence reaction, the enzymatic reaction for
HRP is usually performed before that of AP. This is done because the HRPI
luminol reaction rapidly reaches a steady state light output which is maintained for a short time, then the signal decreases while the AP/dioxetane
reaction slowly reaches the steady-state light emission which then lasts for
a relatively long time. Short washes to remove the HRP substrate are thus
sufficient to avoid any interfering photon emission during the AP reaction. Even if, for reasons of rapidity of the assay, the enzymatic reaction
for HRP is usually performed before that of AP, consistent results can also
be obtained even when the substrate reaction sequence is reversed.
Results
The advantages of chemiluminescence in situ hybridization are mainly:
improved sensitivity, quantification of the data, objectivity of the evaluation and digital imaging of the results.
Improved
sensitivity
Chemiluminescence in situ hybridization proved to be very sensitive,
being able to detect as few as 10 to 50 viral genome copies in infected cells
using both biotynilated probes and digoxigenin labeled probes with HRP
and AP chemiluminescent detections respectively. In fact, positive cherniluminescent signals were obtained in HeLa cells which are known to contain about 10 to 50 integrated genome copies of human papillomavirus
(HPV) 18 (Lorimier et al. 1996, Musiani et al. 1997). Chemiluminescence
in situ hybridization has proved more sensitive than in situ hybridization
followed by colorimetric detection and almost as sensitive as in situ hybridization followed by 35S autoradiography; in fact, the chemiluminescence positivity of the results obtained from the detection of HPV
DNA in HeLa cells was not obtained with colorimetric detection (Lorimier
et al. 1993, Musiani et al. 1997) while chemiluminescent signal resolution
was comparable to that provided by 35S autoradiography (Lorimier et al.
1996). Moreover with chemiluminescence in situ hybridization to detect
B19 parvovirus DNA in several samples of bone marrow cells, all the positive specimens from patients with a diagnosed B19 infection proved positive with a higher number of positive cells/specimen in comparison with