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1151
Micro-Carrier-Test:
Evaluating
Disinfectants
for
HIV
Yukiko SHIMAKOSHI
Second Department of Internal Medicine, Department of Microbiology,
Osaka Medical College, Osaka, Japan
(Received:July 14, 1995)
(Accepted: August 25, 1995)
Key
words:
carrier
test,
HIV,
disinfectant
Abstract
To
of
determine
dry-fixed
cells
the
in
5,ƒÊl
of
flat-bottomed
was
10%
to
remain
cells
(1 •~
and
the
hypochlorite.
discrepancies.
are
Micro-Carrier-Test
to
be
cultured
the
same
the
useful
The
determined
by
reported
previously,
previous
protocols
of
96-well
and
Uninfected
culture
supernatant
transcriptase
cytotoxicity
assay.
was
The
those
a
105
added
PBS.
disinfectants
determined.
(1 •~
of
reverse
glutaraldehyde
screening
well
with
4 weeks.
well-known
and
cells
were
times
0.01%
as
the
each
ethanol,
present
in
of
non-radioisotopic
were
20%
Micro-Carrier-Test
Molt-4
Disinfectants
three
was
several
the
1-infected
bottom
for
by
disinfectants
between
promises
of
the
washed
disinfectant
were
almost
differences
the
the
contact
were
and
I devised
type
temperature.
activity
of
of
of
results
The
inoculated
vitro,
at
room
wells
efficacy
effects
These
the
transcriptase
virucidal
5 minutes
in
virus
dry-fixed
at
and
cytotoxicity
method,
after
were
minutes
were
reverse
time-dependent
concentrations
120
times
cells/well)
viruses
immunodeficiency
serum)
for
designated
Residual
new
against
Human
bovine
measure
week.
the
disinfectants
plate
104
to
every
evaluate
Dose-
fetal
for
harvested
assay
of
cells.
microtiter
allowed
Molt-4
effect
virus-infected
minimal
effective
and
but
0.1%
sodium
there
are
To
reevaluated.
are
some
discussed.
This
disinfectants.
Introduction
Many
(HIV)
investigations
have
been
or
protocol
activity
activity
was
or
infectivity.
viral
virus
can
To
shorten
the
is
sensitive
Viral
to
were
after
7 days
Correspondence
Department
by
the
as
and
to:
the
have
of
dried
Yukiko
of Microbiology,
平 成7年10月20日
previously
with
short-term
step
been
is
is
disinfection.
SHIMAKOSHI,
Medical
to
a
cells
In
the
types
to
these
to
rapid
particles
in
of
the
Carrier
tests
tests
could
5 days12,13).
of
plate.
still
be
Hanson
recovered
et
al.5)
This
which
method
the
other
on
dried
that
2-7 Daigaku-machi,
Takatsuki-shi,
Osaka,
Japan
small
virus
disinfect-
M.D.
College,
is
shorten-
against
stated
a
residual
of
particles
from
the
with
the
method
disinfectants
virus
of
a disinfectant3).
and
other
dry-fixed
if the
locus
in
disinfection
The
enzymes
disinfection
from
a microtiter
disinfectants.
virucidal
viral
Micro-Suspension-Test8),
target
virucidal
disinfectants
according
examine
of
of
of
virus
studies
activity
effect
virus
the
those
HIV
after
two
cells
of
indicator
In
virucidal
the
as
test.
Infectious
the
difficult
uninfected
carrier
accepted
separate
used
immunodeficiency
disinfectants1).
into
It is
efficacy
reported9)11).
HIV-infected
Osaka
divided
introduced
cells
human
components,
of
is needed
co-cultivation
washing
targets
from
we
of
viral
a carrier2)•`7).
1 (HIV-1)-infected
viruses
used
on
are
for
universally
indicator
assays
or
period
major
best
ultracentrifugation
determine
simplifying
disks
infectivity
no
efficacy
of
is the
disinfectants
is
virucidal
antigenicity
a suspension2,3)
type
pathogenic
the
of
there
the
infectivity
determined
enough
and
by
washing
HIV
efficacy
Unfortunately
because
of
infectivity
ing
in
virucidal
determining
Viral
cultured.
tests
suspension
human
for
target
suspension
the
reported.
monitored
be
infectious
of
569
1152
Yukiko
ants
effective
against
Therefore,
a
HIV-infected
of
the
This
step
In
well
of
the
paper,
method
a microtiter
results
of
the
infectants
described.
or
steel,
of
residual
with
the
Cells,
Virus
Molt-4
1-infected
and
and
cells,
cell
them
fetal
ml),
bovine
with
for
(RTA)
in
of
the
X-100)
mixed
and
μl 0f
culture
hundred
was
added
times
the
the
p-nitrophenyl
reaction
was
a
measured
in quadruplicate,
Nacalai
Alcohol
Co.
Ltd.)
distilled
were
96-well
were
fixed
Five
50%
FBS)
as
Drying
of
more
The
the
to
the
the
mean
well
must
assay
are
be
system.
dry-fixed
in the
same
several
in
well.
a
The
well-known
sodium
dis-
(10
after
cells
methanol
at
107
cells/ml
the
The
plate
three
stained
bottom
was
with
of
the
Daiso
Ltd.),
with
was
dried
with
Giemsa
was
for
PBS.
at
37℃.
was
solution.
plate
were
from
2 •~
a
the
(99.9
counted
blank
%
value.
formalde-
v/v
%:
Japan
Pharmaceuticals
saline
residual
an
(PBS)
into
each
time
at
well
cells
were
image
or
room
dry-fixed
experiments
with
was
cutoff
v/v
designated
The
405
The
as
(37
5
nm
inoculated
The
washed
containing
at
Co.).
Sumitomo
FBS)
10
20.
minutes
phosphate-buffered
10%
times
30
ethanol
Hibitane(R):
then
buffer
for
formalin
of
Tween
phosphatase
was
determined
Ltd.),
were
addition
absorbance
InterMed
0.5%
dTTP
the
reaction
the
by
(rA)•E
with
O.02%
well
continued
and
S.D.
%
in
the
third
Japan
%:
diluted
%).
washed
and
v/v
(v/v
plate.
then
was
NaOH,
(5 v/v
being
(2 •~
the
Equipment
gluconate
and
per
a poly
and
by
containing
with
measured
of
streptavidin-alkaline
37•Ž,
of
the
was
(diluted
stopped
ml
transcriptase
dUTP
was
per
streptomycin
well
a sample
buffer
incubation
times
and
each
biotinylated
at
1 N
of
of
Reverse
HIV
in
HIV-
LAV-1BRU12)
supplemented
days.
of
reaction
Persistently
of
77 ƒÊg
4
brief,
containing
of
three
or
50 ƒÊl
(NJ-2001,
hypochlorite
cabinet,
cells
and
and
3
In
cells.
TCID
RPMI-1640
indicator
1 hour
Laboratories
chlorhexidine
in
microliters
50 ƒÊl
2000
washing
buffer
plus
TAAB
the
The
for
of
uninfected
containing
fifty
addition
as
with
plate,
electrophotometer
%:
with
residual
the
was
material
the
cells
of
penicillin
assay14).
with
reaction
microtiter
tightly
on
tests.
every
as
RT)
5 times
hundred
uninfected
biosafety
used
continued
added
of
medium
overnight.
concentrations
flat-bottomed
a
and
fixed
disinfectant
to
performed
maintained
units
buffer
37•Ž
disinfectants
of
in
duplicate.
(LUZEX
and
designated
microliters
temperature
in
used
Conditions
a
Inc.),
to
are
used
microtiter
washed
plate
v/v
Ltd.),
water
Five
of
Tesque
Hanbai
the
infectious
HIV-1-infected
cells
were
the
reaction
second
and
(25
and
concentrations
were
(158
was
at
One
by
microtiter
Glutaraldehyde
cells
of
reaction
phosphate
with
All
first
the
The
stopped
was
transferred
reported
Molt-4
(non-RI
was
buffer.
measured
hyde:
well
washing
was
virus.
Methods
line,
flat-bottomed
the
of
well.
dried
particles3,5)•`7)
material
the
and
assay
and
antibiotics
incubated
microliters
HIV
tonometers,
which
effective
cell
supernatant
Ail of
the
of
infectivity,
previously
infecting
transcriptase
was
and
to
with
The
50
plate
5 MNaCl,
One
and
96-well
and
the
T
three-quarters
reverse
infectious
pipetting,
in
of
by
6 months.
(FBS)
(dT)-immobilized
Triton
2.
over
serum
non-radioisotopic
human
established
replacement
activity
oligo
were
tips
against
of
Disinfectants
established
lines
culturing
10%
Chemical
an
the
infectivity
minimal
results
effective
tests
biohazardous.
Materials
1.
the
or
and
and
compared
on
Micro-Carrier-Test
disinfection
equally
such
studies
or
and
be
many
these
complicated
Micro-Carrier-Test
were
In
scraping
and
to
and
sonication,
the
plate,
assumed
required
determination
by
I describe
be
is
glass
the
surface
makes
this
of
For
the
HIV
been
plates
surface.
from
cannot
for
have
individual
on
removed
HIV
test
cells2,4)•`6)
surface
placed
wet
carrier
SHIMAKOSHI
done
analyzer
III).
microliters
were
of
inoculated
infected
into
cells
each
(10-fold
well
of
dilutions
a 96-well
flat-bottomed
107
to
2 •~
microtiter
104
cells/ml
plate.
感 染症 学 雑 誌
in
The
10,
plate
第69巻
20
or
was
第10号
Carrier
dried
for
120
cells
were
minutes
inoculated
replacement
of
measure
3.
value
of
dry-fixed
,u l of
PBS.
37•Ž
0D405
(Wako
well
of
RT
from
an
every
wells
all-exhausted
cells/200
medium
assay
three
in
(1X104
the
week
type
,u 1/well)
every
was
1153
for HIV Disinfectant
week.
for
and
The
All
with
the
biosafety
incubated
culture
4 weeks.
compared
of
cabinet.
at
37•Ž
in
supernatant
tests
cutoff
were
Uninfected
was
done
in
5%
CO,
with
harvested
to
triplicate.
The
uninfected
cells
value.
Assay
microliters
and
5%
On
react
were
the
Chemical
a disinfectant
to
cells
CO2.
Pure
of
allowed
Uninfected
in
temperature
each
non-RI
hundred
were
at
into
by
Cytotoxicity
One
room
three-quarters
RTA
mean
at
Test
7th
for
was
5 minutes.
inoculated
day
of
Industries
inoculated
The
into
culture,
each
the
into
well
well
viable
each
was
(1
x
cells
then
104
were
well
in
which
washed
cells/200
counted
three
times
Jul/well)
and
with
a Cell
with
150
incubated
Counting
Kit
Ltd.).
Results
1.
Residual
Cells
Residual
air-dried
well
in
after
washing.
the
after
Washing
dry-fixed
cells
a safety
washing.
These
after
cabinet
for
After
findings
30
and
may
washing
60
to
were
120
180
minutes
indicate
counted
minutes,
of
that
with
almost
still
the
100%
air-drying,
wet
or
image
of
almost
the
25%
completely
dried
analyzer
(Fig.
dry-fixed
of
cells
cells
the
cells
are
1) . After
remained
were
easily
being
in
removed
removed
the
by
from
surface.
2.
Survival
The
air-dried
tions
of
was
of
Infectivity
survival
FBS
of
after
infectivity
determined
in
the
(Table
1st
and
Drying
of
2nd
infected
cells
1) . Infectivity
weeks,
and
exposed
of
virus
1
x
to
105
recovery
various
cells/well
was
concentrations
was
highest
in
detected
10%
FBS
of
at
in
all
the
FBS
and
concentra1st
week.
Fig. 1 Residual Cells after Washing
Uninfected cells (1 x 105 cells in 5 ,u1 of 10%
FBS) were air-dried in a 96-well flat-bottomed
microtiter plate for the designated times. After
the drying the wells were washed three times
with PBS. Residual cells were stained with Giemsa solution and analyzed with an image analyzer
(LUZEX III).
Table
1
Survived
Concentrations
*Virus
recovery
OD405 from
平成7年10月20日
three
Infectivity
of Air-dried
Infected
Cells
in Various
of FBS
was
monitored
wells
and
by non -RI RT
the cutoff
value
assay
was
. The
0.161.
value
was
the mean
Yukiko
1154
Table
2
Survived
Infectivity
SHIMAKOSHI
of Air-dried
Infected
*Number of infected cells per well
* *Virus recovery was monitored by reverse transcriptase
(+)
indicates
positive RTA, and (—) negative
Table
3
Effect
Cells in 10% FBS
activity (RTA) .
RTA.
of Disinfectants
*Ethanol , glutaraldehyde,
formalin and sodium hypochlorite
were diluted with PBS and
chlorhexidine gluconate with distilled water to the designated concentrations (v/v %).
**The value was the mean 0D 405from three wells. The cutoff value was 0.170 at the 1st week
and 0.192 at the 2nd week.
***The supernatant of the 3rd and 4th weeks was examined for RTA
, and the residual infectivity
was detected.
感 染 症 学雑 誌
第69巻
第10号
1155
CarrierTestforHIVDisinfectant
To
determine
the
detected,
10-fold
Infectivity
of
rd
week.
detected
3.
Construction
cells
(2
to
remain
for
with
triplicate.
The
was
the
when
1st
4.
mean
the
2nd
was
week,
determined.
week
a
well
was
survival
10%
FBS
and
1
that
x
the
1st
of
were
of
103
1
or
infectivity
could
examined
x
104
lower,
be
(Table
cells/well
2).
in
infectivity
the
could
3
not
the
week
the
the
3rd
week.
for
and
the
each
was
was
well
150
allowed
,u1
of
at
PBS.
37•Ž
supernatant
were
cutoff
to
RTA
If
considered
was
be
the
ineffective,
different
the
to
in
When
was
examined.
in
conducted
value.
considered
the
type
and
incubated
tests
a
When
weeks
flat-bottomed
culture
All
infected
all-exhausted
with
The
was
disinfectant
an
and
with
effective.
4th
in
4 weeks.
of
96-well
times
compared
be
a
in
,u 1/well)
disinfectant
to
weeks,
5.0%
exposure
: 0.5%
and
was
the
and
Effect
of
in
RTA
be
according
formalin;
effect
to
the
-•¢-:
concentrations
the
0.5
after
in
the
effective.
and
0.05%
was
used
chlorhexidine
of
disinfectants
5 minutes
of
were
20%
contact
determined
sodium
0.5%
formalin
after
2, 10,
protocol. -•¡-:
hypochlorite; ------:
and
30
0.05%
and
60
15%
cutoff
sodium
minutes
ethanol;- •~-
contact
3).
At
gluconate,
concentrations
ethanol,
of
ethanol,
(Table
these
Micro-Carrier-Test
0.05%
of
5 minutes
concentrations
Disinfectants
of 15%
virucidal
designated
gluconate
at
5.0,
of
after
concentrations
of
chlorhexidine
effects
concentrations
the
effect
performed
formalin
and
Time-Dependent
critical
virucidal
assay
detected,
hypochlorite,
平 成7年10月20日
was
hypochlorite
cytotoxicity
effective
2
wells
considered
three
every
three
the
sodium
Minimal
medium
of
placed
cells/200
week
of
was
washed
104
every
2nd
was
the
well
microliters
Micro-Carrier-Test
was
of
the
it
then
x
Five
temperature
a disinfectant
(1
devised.
each
room
assay
2 consecutive
glutaraldehyde,
At
and
supernatant
for
the
cytotoxicity
Fig.
from
into
at
was
well
of
Micro-Carrier-Test,
and
1.0%
well
RT
was
minutes
of
each
non-RI
both
the
negative
The
0D405
the
in
formalin,
and
1st
inoculated
120
three-quarters
of
both
of
evaluate
glutaraldehyde,
residual
which
in
the
in
was
for
into
by
value
in
2.0
in
cells/well
in
microliters
time.
of
negative
determined,
102
cells
Micro-Carrier-Test
FBS)
hundred
RTA
it was
was
x
inoculated
dried
inoculated
Characterization
To
1
the
10%
designated
were
positive
and
cells
was
One
measure
supernatant
to
detected
results
in
plate
replacement
to
and
these
The
a
cells
CO2
105
was
of
cells/ml
cabinet.
harvested
x
infected
Micro-Carrier-Test
with
107
Uninfected
1
air-dried
4 weeks.
plate.
biosafety
of
of
cells/well
number
of
x
of
RTA
105
accordance
microtiter
5%
x
the
in
number
dilutions
1
When
be
In
optimum
ethanol,
were
not
0.01%
1156
YukikoSHIMAKOSHI
glutaraldehyde
and 0.1% sodium hypochlorite. Five minutes of contact with 0.5 % formalin or
0.005% chiorhexidine gluconate did not achieve complete disinfection. Their virucidal effect of the
critical concentrations
of 15% ethanol, 0.5 % formalin and 0.05 % sodium hypochlorite was determined at 2, 10, 30 and 60 minutes (Fig. 2) . All these disinfectants showed time-dependent effects in
1 week.
Discussion
In the determination
of the effect of chemical disinfectants
against HIV, carrier tests with
cell-free viruses3'5)-7) and virus-infected cells2'4)^-6)as targets of disinfection have been described.
Hanson et al.5) reported that virus-infected cells were more resistant to disiufectants than cell-free
viruses. When disinfectants are examined only against cell-free viruses, their efficacy for practical
use may be overestimated. Moreover, virus-infected cells are easier to handle than cell-free viruses.
Therefore, virus-infected cells were used as targets of disinfection in the present study. In previous
studies2'-7), the surface of the fixed target was treated with a disinfectant and later it was removed
from the surface by sonication, scraping or pipetting and transferred to a culture system to measure
residual infectivity. This was a complicated and hazardous method. Also some disinfectants, such as
alcohols and aldehydes, may cause the infectious materials to be so tightly attached to the surface
that the target cannot be collected totally and the residual infectivity cannot be measured accurately.
In the present Micro-Carrier-Test
the target is fixed to the bottom of a well in a microtiter plate, and
the treatment with disinfectants and the measurement of residual infectivity can be performed simply
in the same well. With this simple procedure large numbers of samples can be examined safely.
Since the infectivity assay is performed in the same well, residual disinfectant might affect the
results of the assay. The cytotoxicity of the residual disinfectant did indeed affect the monitored cells
in the present test. Aranda-Anzaldo et a1.15)stated that the residual cytotoxicity of the chemical might
mask or mimic the presence of true virucidal activity and lead to erroneous conclusions. Therefore,
a cytotoxicity assay must be performed during the test, and the virucidal effect is not determined at
the concentration of disinfectant which causes residual cytotoxicity.
In order to evaluate the Micro-Carrier-Test,
well-known disinfectants were tested again. Minimal
effective concentrations of ethanol and glutaraldehyde in the Micro-Carrier-Test
were the same as in
the Micro-Suspension-Test.
The minimal effective concentration
of sodium hypochlorite
in the
Micro-Carrier-Test
was slightly higher than in the Micro-Suspension-Test,
probably because of the
greater resistance of dried material. Lloyd-Evans et a1.9) noted that tests conducted on suspensions
often overestimate the ability of a product to disinfect contaminated
surfaces. Hanson et a1.5) also
stated that disinfectants effective against wet HIV cannot be assumed to be equally effective against
dried virus. To avoid overestimation
of virucidal efficacy, carrier tests should be used for the
evaluation of disinfectants.
In carrier tests, 1.1 % glutaraldehyde
for 10 minutes') and 1% for 15 minutes') was found not to
be effective, but the minimal effective concentration in the present test was 0.01% for 5 minutes of
contact. Sattar and Springthorpen noted that virus protected by body fluids may be equally or more
stable. Prince et al.'), Hanson et al.5) and I used 20, 50 and 10% FBS, respectively. So the differences
were probably due to the different concentrations
of FBS.
The virucidal effect of chiorhexidine gluconate could not be examined by the Micro-SuspensionTest previously reported8), but could be by the Micro-Carrier-Test.
Since the cells are destroyed by
low osmotic pressure and low pH, it is difficult to collect the cells by centrifugation
in the
Micro-Suspension-Test.
The present carrier test does not require centrifugation, so chemicals such as
chlorhexidine gluconate can be tested.
感染 症 学 雑 誌
第69巻
第10号
CarrierTestforHIVDisinfectant
1157
The present Micro-Carrier-Test
is a simple procedure which shortens the period of contact
between the infectious target and the disinfectant. There is still the problem that residual disinfectant
can affect subsequent co-cultivation. However, the use of the cytotoxicity assay solves this problem.
Accordingly, both the previous Micro-Suspension-Test
and the present Micro-Carrier-Test
are
required in the screening of disinfectants.
Acknowledgments
Grateful acknowledgments
are made to Prof. Masuyo Nakai,
to Prof. Ken-ichi Katsu, Second Department of Internal Medicine
wish to express my thanks to Dr. Kouichi Sano for helpful support
Ms. Akie Hanada for their expert technical assistance.
Part of this work was supported by a grant for AIDS Research
Public Health.
Department of Microbiology and
for their guidance in this study. I
and to Mr. Yoshihiko Fujioka and
from The Osaka Association
for
References
1) Sattar, S.A. and Springthorpe, V.S.: Survival and disinfectant inactivation of the human immunodeficiency virus:
A critical review. Rev. Infect. Dis. 13: 430-447, 1991.
2) Resnick, L., Veren, K., Salahuddin, S.Z., Tondreau, S. and Markham, P.D.: Stability and inactivation of HTLV-III/
LAV under clinical and laboratory environments. JAMA 255(14): 1887-1891, 1986.
3) Tj cktta, E., Hungnes, 0. and Grinde, B.: Survival of HIV-1 activity after disinfection, temperature and pH changes,
or drying. J. Med. Virol. 35: 223-227, 1991.
4) Fauvel, M. and Ozanne, G.: Immunofluorescence assay for human immunodeficiency virus antibody: Investigation
of cell fixation for virus inactivation and antigen preservation. J. Clin. Microbiol. 27(8): 1810-1813, 1989.
5) Hanson, P.J.V., Gor, D., Jeffries, D.J. and Collins, J.V.: Chemical inactivation of HIV on surfaces. BMJ 298:
862-864, 1989.
6) Pepose, J.S., Linette, G., Lee, S.F. and MacRae, S.: Disinfection of Goldmann tonometers against human immunodeficiency virus type 1. Arch. Ophthalmol. 107: 983-985, 1989.
7) Prince, D.L., Prince, R.N. and Prince, H.N.: Inactivation of human immunodeficiency virus type 1 and herpes
simplex virus type 2 by commercial hospital disinfectants. Chemical TIMES & TRENDS 13: 13-16, 1990.
8) Shimakoshi, Y., Sano, K., Nakano, T., Nakamura, T., Ohshiba, S., Katsu, K. and Nakai, M.: A micro-suspensiontest for evaluation of disinfectants against human immunodeficiency virus. J. Jpn. Assoc. Infect. Dis. 69(5):
532-538, 1995.
9) Lloyd-Evans, N., Springthorpe, V.S. and Sattar, S.A.: Chemical disinfection of human rotavirus-contaminated
inanimate surfaces. J. Hyg. 97: 163-173, 1986.
10) Tyler, R. and Ayliffe, G.A.J.: A surface test for virucidal activity of disinfectants: preliminary study with herpes
virus. J. Hosp. Infect. 9: 22-29, 1987.
11) Sattar, S.A., Springthorpe, V.S., Karim, Y. and Loro, P.: Chemical disinfection of non-porous inanimate surfaces
experimentally contaminated with four human pathogenic viruses. Epidem. Inf. 102: 493-505, 1989.
12) Barre-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C.,
Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Montagnier, L.: Isolation of a T-lymphotropic retrovirus from
a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220:868-871, 1983.
13) Bueren, J., Simpson, R.A., Jacobs, P. and Cookson, B.D.: Survival of human immunodeficiency virus in suspension
and dried onto surfaces. J. Clin. Microbiol. 32(2):571-574, 1994.
14) Nakano, T., Sano, K., Odawara, F., Saitoh, Y., Otake, T., Nakamura, T., Hayashi, K., Misaki, H. and Nakai, M.:
An improved non-radioisotopic reverse transcriptase assay and its evaluation. J. Jpn. Assoc. Infect. Dis. 68(7):
923-931, 1994.
15) Aranda-Anzaldo, A., Viza, D. and Busnel, R.G.: Chemical inactivation of human immunodeficiency virus in vitro.
J. Virol. Methods 37: 71-82, 1992.
平成7年10月20日
1158
Yukiko
Micro-Carrier-Test:
SHIMAKOSHI
HIVに
対 す る消 毒 薬 の 評 価 方 法
大阪医科大学 第2内 科学教室,微 生物学教室
島
要
越
assayで 残 存 す る消 毒 薬 の細 胞 毒 性 を検 討 した.
旨
ウイル ス に対 す る消 毒薬 の効 果判 定 方 法 とし
て,乾
燥 固 定 した ウ イ ル ス 感 染 細 胞 を用 い た
Micro-Carrier-Testを
イ ル ス1型
考 案 し た.ヒ
ト免 疫 不 全 ウ
に 感 染 し たMolt44を96穴
平 底 マ イ ク
ロ タ イ タ ー プ レ ー トの ウ エ ル 底 に 室 温 で120分
乾 燥 固 定 し 消 毒 薬 を 作 用 さ せ た 後,PBSで
し,非 感 染 のMolt-4を
加 え4週
由紀 子
間
洗浄
RT
assayで
転 写 酵 素 活 性 を 測 定 し た.ま
たcytotoxicity
果 を再 検 討 した.濃 度,時
られ,5分
ル,0.01%グ
間依存性 の効果 が認 め
間 で の最 低 有効 濃 度 は,20%エ
ル タル ア ル デ ヒ ド,0.1%次
タ ノー
亜 塩素酸
ナ トリウ ム で あ っ た.こ れ は,こ れ ま で の 報 告 と
部 違 い が あ っ た の で,今
回 と これ ま で の 方 法 に
一
つ い て そ の 違 い を検 討 し た.こ のMicro-Carrier-
間 培 養 し た.1週
間 毎 に 培 養 上 清 を 回 収 しnon-RI
この 新 しい 方 法 を評 価 す るた め既 存 の 消 毒 薬 の 効
逆
Testは
消 毒 薬 の効 果 判 定 の 一 つ の ス ク リー ニ ン
グ法 と して 有 効 で あ る と考 え る.
感 染症 学 雑誌
第69巻
第10号