Microbiol. Immunol. Vol. 21 (10), 583-591, 1977
A Simple
Hideo
and Efficient Microassay for Titration of Interferon
ISHITSUKA,
Yasuko
NOMURA,
and
Method
Kouichi
TAKANO
Departmentof Microbiologyand Chemotherapy,Nippon RocheResearch Center, Kanagawa (Received for publication, May 13, 1977)
Abstract A simple and efficient microassay method for the titration of interferon was developed by the use of microtest plates for handling a large number of samples. L929 cells pretreated with interferon were infected with vesicular stomatitits virus (VSV) and cultured in the presence of 3H-uridine. The activity was expressed by the reduction of extracellular radioactive RNA released after destruction of the infected cells, which was measured in terms of the radioactivity incorporated into cold TCA-insoluble materials in the culture fluid. The interferon titer determined by this method was in the same order as that by the plaque reduction method. The activity by this method was parallel to, but lower than that expressed by the yield reduction of infectious viruses. This method requires only 0.025 ml of each test sample with higher than 1 NIH ref. unit/ml to detect its interferon activity and takes 2 to 3 days for assaying hundreds of samples.
Efforts have been made to develop a rapid, simple, and convenient microassay method for interferon for the purpose of screening a large number of microbial products for interferon inducers. Interferon has been assayed in terms of its ability to inhibit the growth of viruses. Many different assay methods have been developed, based on various ways of measuring viral growth (3). Among them the radiochemical assay, in which the activity was expressed by the degree of inhibition of radioactivity from being incorporated into viral RNA in the infected cells, was the most rapid, convenient, and reproducible method, although this is far from having the simplicity to be emphasized (1, 3, 4). In this communication a radiochemical assay method based on another principle is described which is simpler and more efficient for assaying a large number of samples. MATERIALS
Cell medium 100
cultures. (MEM)
,ƒÊg/ml
cultured
L929
streptomycin in
microtest
mouse
containing
fibroblasts 10%
and plates
AND
20 for
calf units/ml
tissue
METHODS
were serum,
cultured 5%
penicillin. culture 583
(Linbro).
tryptose For
with
minimum
phosphate interferon
essential broth
assay,
(TPB), cells
were
584
H.
Viruses. virus
Vesicular
were
stored
and Interferon
in
microtest
of
a
by
test the
use
which
of
layers
of
sample
ple
the L929 in
in
the
were added
fluid
in
If
samples
well
ferred
onto
a
paper
discs
with
Fig.
the
with
fluid
of
serotype) L929
and
cells.
Mengo
Viruses
after
paper
disc
culture
Illustrative
(Whatman fluid
piled
were
L929
explanation
After
an
in
0.1
calf
were
hole
cultured Mengo
an
5%
cold
of the radiochemical
and
TCA
were 96
where
plate
was
lifted
and
mixed
into
an
or
the pin in
microassay
in a
plate monooff,
the
culture sam-
3H-uridine
the
cells,
wells,
interferon
VSV.
ml
made
transfer
plate,
culture,
insect
cultured 0.025
having The
with
from
with
were
dilutions
virus
overnight
units/mg.
ml)
plate
transfer
infected. precipi-
ref.
serum
Co.).
small
released
3MM)
in
a microtest
cells with
RNA,
placed
cells
a transfer
the
cells sulfate
NIH
serially,
onto
the
106
5%
in
When
infection.
104
Engineering
challenged
radioactive
1.36 •~
diluted
Lab.)
through plate.
and
be
(Cook
cultured.
microtest MEM
(6 •~
L929
ammonium
containing to
was
mouse by
was
cells
(Flow apexes
from purified
MEM
needed
transferred
containing
1.
L929
sample
been
was
immediately a well
the
diluted had
of
were
at
was activity
in
Multidiluter
serially
well
of
C overnight
holes
washed
was
37
cells
each
Jersey
culture
prepared
and
Specific
Monolayers at
minor
the
was
(NDV) (2).
a Titertek
have
containing
fluid
assay.
sample.
(VSV-New
from
interferon virus
Sephadex
plates
ET AL
use.
disease CM
virus
prepared
Murine
Newcastle
tation
and
C until
Interferon. with
stomatitis
harvested
at -70
ISHITSUKA
was the
beaker
method.
supernatant then
trans-
center. for
The 10
min.
MICROASSAY
METHOD
FOR
585
INTERFERON
Then the discs were washed with 5% cold TCA 3 times and dehydrated with ethanol and ether in the same beaker. Cold TCA-insoluble fractions on the paper discs were counted in a liquid scintillation counter. A scheme of this assay method is illustrated in Fig. 1. Chemical. 3H-uridine (5T, 19.7 Ci/mmole) was purchased from Daiichi Pure Chemicals Co. RESULTS
Incorporationof 3H-uridineand Releaseof RadioactiveRNA from the InfectedCells Monolayers of mouse L929 cells were infected with VSV and cultured in the presence of 3H-uridine. The present method is based on the following principle. 3H-uridine is considered to be incorporated into viral and cellular RNAs . If the cells are destroyed after viral replication, the intracellular contents including viral and cellular RNAs would be released into the culture fluid. The experiment illustrated in Fig. 2 confirmed the above rationale. In this experiment, the infected cells were cultured with or without actinomycin D in the presence of 3H-uridine. After an overnight culture, 3H-RNA released into the culture fluid was fractionated by sucrose density gradient centrifugation. As shown in the figure, RNA in viral particles together with cellular ribosomal RNA and a lower molecular weight RNA fraction were observed. In the culture with actinomycin D, cellular ribosomal
Fig. 2. Patterns of cold TCA-insolubles released into culture fluid in VSV-infected cells. Monolayers of L-929 cells were infected with VSV at an MOI of 5 and cultured with or without actinomycin D at 0.2 was added to the culture μg/ml for 17 hr. 3H-uridine at
1 hr
μCi/ml. was put and for
was 90
insolubles with
after viral After the on
infection culture
sucrose
density
centrifuged min.
at
Fractions in
actinomycin
0.1
ml
at a concentration 1 ml of the culture gradient
26,000 were
of
D; •œ,
each
(15-40%,
rpm
in
pooled fraction
without
an and
were actinomycin
of 0.5 fluid 35
SW27
ml) rotor
cold
TCA-
counted. •›, D.
586
IL ISHITSUKA
Fig.
3.
Incorporation
insolubles cells
(6
with
cold
counted
0.5;
A,
cell
cells) at
hr
after
Concentrations
3H-uridine
culture in
various
TCA-isolubles 24
text.
of
the
104
VSV
The
in
in
a
ET AL
fluid.
into
microtest
plate
concentrations in
culture
incubation. (ƒÊCi/ml)
cold
Monolayers were of fluids Details : •›,
0.125;•œ
infected
3H-uridine.
(0.05 are
TCAof L929
ml)
were
described 0.25; •¢,
1.0.
3H-RNA disappeared and most of the lower molecular weight RNA fraction remained, suggesting that most of the latter RNA fraction was viral RNA. Since the above data showed the release of radioactivity into the culture fluid not only of viral but also of cellular RNA, it was suggested that the release was due to destruction of the infected cells. Figure 3 shows the effect of multiplicity of infection (MOI) and the concentration of 3H-uridine on the release of cold TCA-insoluble counts. The counts were dependent on the concentration of the labeled compound in the range of MOI between 1.25 and 20. Influenceof Interferonon the Releaseof RadioactiveRNA Influence of interferon on the destruction of the infected cells was investigated, which was represented by the release of 3H-labeled RNA into the culture fluid. Extracellular radioactive RNA measured by counts of the cold TCA-insoluble fraction in the culture fluid increased until 24 hr and this increase was almost completely inhibited when the cells were pretreated with 100 units/ml interferon (Fig. 4). Table 1 shows the distribution of 3H-uridine incorporated into RNA among intracellular and extracellular parts. In the uninfected culture, 3H-uridine was incorporated into intracellular RNA and release of the radioactivity into the culture fluid was not observed. On the other hand, in VSV infected culture, the release of the radioactivities from the cells to the culture fluid was observed. Interferon pretreatment inhibited the release of the radioactive RNA from the infected cells. These results indicated that interferon inhibited the destruction of the infected cells.
MICROASSAY Table
1.
L929
cells, for
an
for
hr.
culture
which
24
MOI
17
FOR
587
INTERFERON
Distribution of 3H-uridine incorporated into among intracellular and extracellular parts
terferon at
METHOD
hr
in
of 5, After
fluid
were
and
the and
pretreated
a microtest cultured
culture, in
with cold
the
cells
with
plate,
were
or
were
RNA
without
infected
3H-uridine
in-
by
VSV
(0.5 ƒÊCi/ml)
TCA-insoluble
counts
in
the
measured.
(B )
(A )
Fig.
5.
Dose-response
inhibition
interferon-pretreated in
Fig.
4.
Growth
lular ed
cell
are
illustrated
cells in
and
radioactive cultures.
Two
with
and
further
uridine
infection,
the
cell
the
culture
ted.
(A) •›,
10
nuits/ml
ture.
(B) •›,
100
units/ml
ture.
free
in
the
At cold
fluids
untreated
from
plate 10
of interferon. VSV at an
cultured
(A)
or
various
100
of 3H-
times
after in
(0.05
ml)
were
L929
cell
culture; •œ,
cell
culture; •œ,
L929
interferon-pretreated
coun-
cell
cell
plates
concentrations
hr.
cells
The
cultured
in
pCilml)
for
the
TCA
cold ml)
the
tures tion
into
of
cul-
cold
with
plaques ble C(•›),
100
were
B(•¢),
extracellular
and
the
of
viral virus
the cul-
incorpora (A)
of VSV in dishes
after
of
by
Control
of
into
yield
(C).
and
(C). B: (6 cm)
concentrations
The
PFU
fluids
dpm
various hr.
counted
incorporation counts;
16
of
culture
TCA-insolubles
with for
16 and (0.5
assayed
107 PFU/ml L929 cells
pretreated
A(•œ),
(A)
5,830
9.8 •~ of
interferon
fected cul-
the
VSV
radioactivity the
was
method
produced Monolayers were
the
counted
represented
with for
with
in
virus
plaque-forming
cells
of 3H-uridine
Then
insolubles
were
in
L929
interferon
infected
hr.
C:
pretreated
of
presence
24
curve
A
were
were
extracellular
cells of 20
TCA-insolubles
microtest
various
(0.05
were
The MOI
presence
interferon-pretreated untreated
of L929
a microtest
(1.0 ƒÊCi/m1).
the
experiments
Monolayers
or
ref. units/ml infected with
extracel-
terferon-treat-
separate
in
(B) were
of in
together.
(6 •~ 104 cells)
contact
inhibition
virusesin
cells.
cells VSV 3 days'
cold plaque yield.
were and
inviral
culture.
TCA-insolureduction;
588
H.
ISHITSUKA
ET AL
Titration of InterferonActivityby the Inhibition of the Releaseof 3H-RNA L929 cells pretreated with various concentrations of interferon were infected with VSV and cultured in the presence of 3H-uridine. Twenty-four hours after infection the cold TCA-insoluble counts in the culture fluid, which represented the radioactive RNA released by lysis of infected cells, were measured. As shown in Fig. 5 (A), the radioactivity was found to be related to the concentration of interferon as indicated by an S-shaped curve which appeared linear in the range corresponding to about 20-75% of radioactivity in the control, or to about 0.25-3 NIH ref. units/ml of interferon. This means that the interferon activity could be reliably detected with 0.1 ml of a sample in the well having more than 0.25 unit/ml of interferon. The concentration of an interferon preparation at which the radioactivity was 50% that in the control was arbitrarily taken as 1 unit/ml, which represented about 0.6 NIH ref. unit/mi. Interferon activity assayed by this method was compared with that assayed by other methods as shown in Fig. 5 (A, B, and C). The activity expressed by this radiochemical microassay method was parallel to, but lower than that expressed by yield reduction of extracellular infectious viruses (A, C). The titer of interferon assayed by this method and that determined by the usual plaque reduction method also seemed to be within the same range (A, B).
Fig.
6.
Time
of
quired for Monolayers plate were ref. units/ml Then
the
contact
in cells
MOI of 20 3H-uridine
the cells
contact with of interferon were
and cultured (1 .0 ,ƒÊCi/ml) counts
were
measured
and
age
of control.
Control
interferon
with in
plotted
VSV
the 24
the
at
culture as the
represented
The
of cold fluids
percent13,056
7.
viruses
an
presence hr.
Fig.
Growth
and
extracellular
1.2, 12 and 120 for various times.
for in
re-
antiviral state. in the microtest
infected
TCA-insoluble
dpm.
with
obtaining of L929
in
L cells
untreated
with
units/ml) cultured
for
Details
at
an
MOI
various
described
left
(10
infected
fluids are
or
interferon
were virus
cell
pretreated
ref. with
of 20
and
in
the
times
presence of 3H-uridine The cold TCA-insolubles culture
of Mengo
interferon-treated
cultures.
Mengo
inhibition
radioactive
(1,ƒÊCi/ml). in the
were
counted. in
the
text.
MICROASSAY
METHOD
FOR
589
INTERFERON
Time of PretreatmentRequiredfor the Cells to Obtain the State of Viral Resistance It is known that the synthesis of the antiviral protein and development of the antiviral state occur rapidly within 2 hr and reach a maximum somewhere between 5 and 8 hr (5) following the contact of cells with interferon. In this radiochemical microassay method, suggestive results were obtained as shown in Fig. 6. At various times of contact of L929 cells with interferon, the cells were infected with VSV, and cold TCA-insolubles in the culture fluid were counted 24 hr after infection. Figure 6 shows that 2 hr contact of the cells with interferon was sufficient in this assay for detecting the relative antiviral activity, and a maximum antiviral state of cells was obtained 8 hr following contact with interferon.
Fig.
8.
Inhibition
dose-response
interferon
of Mengo
viruses
pretreated trations
overnight were infected
with
presence insoluble and
the
of
3H-uridine
counts counts
and
in are
and infecting interferon cultured
(0.51ƒÊCi/ml). the plotted
culture as the
yield-reduction L cells. at
L929
by cells
various concenfor 17 hr in the The
fluids percentage
were
cold
TCA-
measured of control.
Release of Radioactive RNA in L Cell-Mengo Virus System This radiochemical microassay method was employed in another viral infection system and showed a similar tendency. Figure 7 illustrates extracellular RNA released from the infected cells in terms of the incorporation of 3H-uridine into cold TCA-insoluble counts of the culture fluid. Extracellular radioactive RNA was detected as early as 10 hr after infection and continued to increase up to 14 hr. In interferon-treated cells the release of radioactive RNA was clearly inhibited. Figure 8 shows the dose-response inhibition curve in Mengo virus infection obtianed by this assay method. DISCUSSION
Allen and Giron (1) developed an assay method for interferon, in which they assessed the growth of MM virus in terms of the amount of 3H-uridine incorporated
590
H.
into
viral
RNA.
containing the
synthesis
of
containing
this
and
trapped
cellular
cold were
is
rapid
and
in
which
synthesis
in
infected
On
the
other
of
feron
preparations
ously.
The
by
TCA,
cold
Although to
simpler
Assays
and of
with
interferon
virus
may The
existing the
for microtest
yield the
of titers virus
the
same
the
be
when
a
could
be
a
the the
1 hr
yield use
with
extracellular of interferon almost
We thank Drs. M. Iizuka their kind supply of NIH
was
same
to
the
this method radiochemical
inhibition
to
of
viral
in
RNA
with
3H-uridine to
a
which
caused
preincubated
by inter-
simultane-
filter
paper,
were
requires
a
synthesis
fixed
washed
and
shorter
time
than
precedes
the
release
microassay number
of
method samples
within
based the
pancreatic in
3H-labeled
the
compared
radioactivity
RNA
2 to
more
rapid
on
the
inhibition
our of
proposed
are
3 days
a
microassay
of radioactive treatment.
except
assayed
me-
1).
for
however
of
RNAs
buffered
to
here
be
after
assayed.
the
contact
operation,
Mengo
virus.
reduction of RNase
addition
plate,
was
cells,
into
method,
with
with
large
method
infected
as by
added transferred
completed
challenge
microassay the
and
Furthermore, as
as
by
cells
was
radiochemical
efficient
medium
Radioactivity
radioactive
The
method
of samples
used
of
(Fig.
inhibit
with
simplicity.
virus
viral
pads.
microassay
papers
medium to
above-mentioned
expressed
RNA
because
cells,
fiber
have modified cell system, the
in
release
beaker
later
scraped
simplicity (4)
is
simple.
filter
were
the culture
3 hr
The
from
lack
of
radiochemical
the
cells glass
et al VSV-L
to
each
2 to
radiochemical
of
results
from
present of
cultured
for
the
in
far
activity
with
preparation.
principle such same manner
progeny
time
more
well
destruction
here,
one
is
seems
radioactive hundreds
on
infected,
to
ethanol.
Suzuki with a
is very
infected
the
hundreds
it
were later
the
trapped
inhibition
cells,
and
RNA
infection,
present
by
dishes min
replaced
with
still
the
in 30
was
interferon
cells,
infected
obtain
radioactive
after
however,
were
cells
fluid
Although more rapid
expressed
the
at
method
the
were
the
ET AL
added
dehydration
extracellular
dehydrated
the
hours
hand,
was
destruction
the
Six
efficient,
method,
in
and
method. and
RNA
is
RNA
after
assay
activity
L929 D was
TCA-insolubles counted
plaque reduction be more convenient
the
method,
actinomycin
3H-uridine.
saline
thod
In
5 ,ƒÊg/ml
ISHITSUKA
RNase
virus the
as
that
counted.
by
and S. Kobayashi reference interferon.
at
viruses
yield
in Basic
would
Research
of the
of
are
extracellular method
Laboratory,
in the further 10 ƒÊg/ml
digested
data
of
another
be devised cells were
be
Although
by
on
a concentration
reduction
obtained
interferon
based
viruses could the infected
If
progeny
by
method
and not
only shown
3H-labeled mentioned
Toray
Industries,
here. Inc.
REFERENCES
1) 2) 3)
Allen, P.T., and Giron, D.J. 1970. Rapid, sensitive assay for interferons based on the inhibition of MM virus nucleic acid synthesis. Appl. Microbiol. 20: 317. Fantes, K.H. 1966. Purification, concentration and physicochemical properties of interferons, p. 119. In Finter, N.B. (ed), Interferons, North-Holland Publishing Co., Amsterdam. Finter, N.B. 1973. The assay and standardization of interferon and interferon inducers, p. 135. In Finter, N.B. (ed), Interferon and interferon inducers, North-Holland Publishing Co., Amsterdam.
MICROASSAY
4) 5)
METHOD
FOR
INTERFERON
591
Suzuki, J., Akaboshi, T., and Kobayashi, S. 1974. A rapid and simple method for assaying interferon. Japan. J. Microbiol. 18: 449. Wagner, R.R. 1961. Biological studies of interferon. I. Suppression of cellularinfection with Eastern equine encephalomyelitis virus. Virology 13: 323.
Requests for reprints should be addressed to Dr. H. Ishitsuka, Department of Microbiology and Chemotherapy, Nippon Roche Research Center, 200 Kajiwara, Kamakura-city, Kanagawa, Japan.
A Simple
Hideo
and Efficient Microassay for Titration of Interferon
ISHITSUKA,
Yasuko
NOMURA,
and
Method
Kouichi
TAKANO
Departmentof Microbiologyand Chemotherapy,Nippon RocheResearch Center, Kanagawa (Received for publication, May 13, 1977)
Abstract A simple and efficient microassay method for the titration of interferon was developed by the use of microtest plates for handling a large number of samples. L929 cells pretreated with interferon were infected with vesicular stomatitits virus (VSV) and cultured in the presence of 3H-uridine. The activity was expressed by the reduction of extracellular radioactive RNA released after destruction of the infected cells, which was measured in terms of the radioactivity incorporated into cold TCA-insoluble materials in the culture fluid. The interferon titer determined by this method was in the same order as that by the plaque reduction method. The activity by this method was parallel to, but lower than that expressed by the yield reduction of infectious viruses. This method requires only 0.025 ml of each test sample with higher than 1 NIH ref. unit/ml to detect its interferon activity and takes 2 to 3 days for assaying hundreds of samples.
Efforts have been made to develop a rapid, simple, and convenient microassay method for interferon for the purpose of screening a large number of microbial products for interferon inducers. Interferon has been assayed in terms of its ability to inhibit the growth of viruses. Many different assay methods have been developed, based on various ways of measuring viral growth (3). Among them the radiochemical assay, in which the activity was expressed by the degree of inhibition of radioactivity from being incorporated into viral RNA in the infected cells, was the most rapid, convenient, and reproducible method, although this is far from having the simplicity to be emphasized (1, 3, 4). In this communication a radiochemical assay method based on another principle is described which is simpler and more efficient for assaying a large number of samples. MATERIALS
Cell medium 100
cultures. (MEM)
,ƒÊg/ml
cultured
L929
streptomycin in
microtest
mouse
containing
fibroblasts 10%
and plates
AND
20 for
calf units/ml
tissue
METHODS
were serum,
cultured 5%
penicillin. culture 583
(Linbro).
tryptose For
with
minimum
phosphate interferon
essential broth
assay,
(TPB), cells
were
584
H.
Viruses. virus
Vesicular
were
stored
and Interferon
in
microtest
of
a
by
test the
use
which
of
layers
of
sample
ple
the L929 in
in
the
were added
fluid
in
If
samples
well
ferred
onto
a
paper
discs
with
Fig.
the
with
fluid
of
serotype) L929
and
cells.
Mengo
Viruses
after
paper
disc
culture
Illustrative
(Whatman fluid
piled
were
L929
explanation
After
an
in
0.1
calf
were
hole
cultured Mengo
an
5%
cold
of the radiochemical
and
TCA
were 96
where
plate
was
lifted
and
mixed
into
an
or
the pin in
microassay
in a
plate monooff,
the
culture sam-
3H-uridine
the
cells,
wells,
interferon
VSV.
ml
made
transfer
plate,
culture,
insect
cultured 0.025
having The
with
from
with
were
dilutions
virus
overnight
units/mg.
ml)
plate
transfer
infected. precipi-
ref.
serum
Co.).
small
released
3MM)
in
a microtest
cells with
RNA,
placed
cells
a transfer
the
cells sulfate
NIH
serially,
onto
the
106
5%
in
When
infection.
104
Engineering
challenged
radioactive
1.36 •~
diluted
Lab.)
through plate.
and
be
(Cook
cultured.
microtest MEM
(6 •~
L929
ammonium
containing to
was
mouse by
was
cells
(Flow apexes
from purified
MEM
needed
transferred
containing
1.
L929
sample
been
was
immediately a well
the
diluted had
of
were
at
was activity
in
Multidiluter
serially
well
of
C overnight
holes
washed
was
37
cells
each
Jersey
culture
prepared
and
Specific
Monolayers at
minor
the
was
(NDV) (2).
a Titertek
have
containing
fluid
assay.
sample.
(VSV-New
from
interferon virus
Sephadex
plates
ET AL
use.
disease CM
virus
prepared
Murine
Newcastle
tation
and
C until
Interferon. with
stomatitis
harvested
at -70
ISHITSUKA
was the
beaker
method.
supernatant then
trans-
center. for
The 10
min.
MICROASSAY
METHOD
FOR
585
INTERFERON
Then the discs were washed with 5% cold TCA 3 times and dehydrated with ethanol and ether in the same beaker. Cold TCA-insoluble fractions on the paper discs were counted in a liquid scintillation counter. A scheme of this assay method is illustrated in Fig. 1. Chemical. 3H-uridine (5T, 19.7 Ci/mmole) was purchased from Daiichi Pure Chemicals Co. RESULTS
Incorporationof 3H-uridineand Releaseof RadioactiveRNA from the InfectedCells Monolayers of mouse L929 cells were infected with VSV and cultured in the presence of 3H-uridine. The present method is based on the following principle. 3H-uridine is considered to be incorporated into viral and cellular RNAs . If the cells are destroyed after viral replication, the intracellular contents including viral and cellular RNAs would be released into the culture fluid. The experiment illustrated in Fig. 2 confirmed the above rationale. In this experiment, the infected cells were cultured with or without actinomycin D in the presence of 3H-uridine. After an overnight culture, 3H-RNA released into the culture fluid was fractionated by sucrose density gradient centrifugation. As shown in the figure, RNA in viral particles together with cellular ribosomal RNA and a lower molecular weight RNA fraction were observed. In the culture with actinomycin D, cellular ribosomal
Fig. 2. Patterns of cold TCA-insolubles released into culture fluid in VSV-infected cells. Monolayers of L-929 cells were infected with VSV at an MOI of 5 and cultured with or without actinomycin D at 0.2 was added to the culture μg/ml for 17 hr. 3H-uridine at
1 hr
μCi/ml. was put and for
was 90
insolubles with
after viral After the on
infection culture
sucrose
density
centrifuged min.
at
Fractions in
actinomycin
0.1
ml
at a concentration 1 ml of the culture gradient
26,000 were
of
D; •œ,
each
(15-40%,
rpm
in
pooled fraction
without
an and
were actinomycin
of 0.5 fluid 35
SW27
ml) rotor
cold
TCA-
counted. •›, D.
586
IL ISHITSUKA
Fig.
3.
Incorporation
insolubles cells
(6
with
cold
counted
0.5;
A,
cell
cells) at
hr
after
Concentrations
3H-uridine
culture in
various
TCA-isolubles 24
text.
of
the
104
VSV
The
in
in
a
ET AL
fluid.
into
microtest
plate
concentrations in
culture
incubation. (ƒÊCi/ml)
cold
Monolayers were of fluids Details : •›,
0.125;•œ
infected
3H-uridine.
(0.05 are
TCAof L929
ml)
were
described 0.25; •¢,
1.0.
3H-RNA disappeared and most of the lower molecular weight RNA fraction remained, suggesting that most of the latter RNA fraction was viral RNA. Since the above data showed the release of radioactivity into the culture fluid not only of viral but also of cellular RNA, it was suggested that the release was due to destruction of the infected cells. Figure 3 shows the effect of multiplicity of infection (MOI) and the concentration of 3H-uridine on the release of cold TCA-insoluble counts. The counts were dependent on the concentration of the labeled compound in the range of MOI between 1.25 and 20. Influenceof Interferonon the Releaseof RadioactiveRNA Influence of interferon on the destruction of the infected cells was investigated, which was represented by the release of 3H-labeled RNA into the culture fluid. Extracellular radioactive RNA measured by counts of the cold TCA-insoluble fraction in the culture fluid increased until 24 hr and this increase was almost completely inhibited when the cells were pretreated with 100 units/ml interferon (Fig. 4). Table 1 shows the distribution of 3H-uridine incorporated into RNA among intracellular and extracellular parts. In the uninfected culture, 3H-uridine was incorporated into intracellular RNA and release of the radioactivity into the culture fluid was not observed. On the other hand, in VSV infected culture, the release of the radioactivities from the cells to the culture fluid was observed. Interferon pretreatment inhibited the release of the radioactive RNA from the infected cells. These results indicated that interferon inhibited the destruction of the infected cells.
MICROASSAY Table
1.
L929
cells, for
an
for
hr.
culture
which
24
MOI
17
FOR
587
INTERFERON
Distribution of 3H-uridine incorporated into among intracellular and extracellular parts
terferon at
METHOD
hr
in
of 5, After
fluid
were
and
the and
pretreated
a microtest cultured
culture, in
with cold
the
cells
with
plate,
were
or
were
RNA
without
infected
3H-uridine
in-
by
VSV
(0.5 ƒÊCi/ml)
TCA-insoluble
counts
in
the
measured.
(B )
(A )
Fig.
5.
Dose-response
inhibition
interferon-pretreated in
Fig.
4.
Growth
lular ed
cell
are
illustrated
cells in
and
radioactive cultures.
Two
with
and
further
uridine
infection,
the
cell
the
culture
ted.
(A) •›,
10
nuits/ml
ture.
(B) •›,
100
units/ml
ture.
free
in
the
At cold
fluids
untreated
from
plate 10
of interferon. VSV at an
cultured
(A)
or
various
100
of 3H-
times
after in
(0.05
ml)
were
L929
cell
culture; •œ,
cell
culture; •œ,
L929
interferon-pretreated
coun-
cell
cell
plates
concentrations
hr.
cells
The
cultured
in
pCilml)
for
the
TCA
cold ml)
the
tures tion
into
of
cul-
cold
with
plaques ble C(•›),
100
were
B(•¢),
extracellular
and
the
of
viral virus
the cul-
incorpora (A)
of VSV in dishes
after
of
by
Control
of
into
yield
(C).
and
(C). B: (6 cm)
concentrations
The
PFU
fluids
dpm
various hr.
counted
incorporation counts;
16
of
culture
TCA-insolubles
with for
16 and (0.5
assayed
107 PFU/ml L929 cells
pretreated
A(•œ),
(A)
5,830
9.8 •~ of
interferon
fected cul-
the
VSV
radioactivity the
was
method
produced Monolayers were
the
counted
represented
with for
with
in
virus
plaque-forming
cells
of 3H-uridine
Then
insolubles
were
in
L929
interferon
infected
hr.
C:
pretreated
of
presence
24
curve
A
were
were
extracellular
cells of 20
TCA-insolubles
microtest
various
(0.05
were
The MOI
presence
interferon-pretreated untreated
of L929
a microtest
(1.0 ƒÊCi/m1).
the
experiments
Monolayers
or
ref. units/ml infected with
extracel-
terferon-treat-
separate
in
(B) were
of in
together.
(6 •~ 104 cells)
contact
inhibition
virusesin
cells.
cells VSV 3 days'
cold plaque yield.
were and
inviral
culture.
TCA-insolureduction;
588
H.
ISHITSUKA
ET AL
Titration of InterferonActivityby the Inhibition of the Releaseof 3H-RNA L929 cells pretreated with various concentrations of interferon were infected with VSV and cultured in the presence of 3H-uridine. Twenty-four hours after infection the cold TCA-insoluble counts in the culture fluid, which represented the radioactive RNA released by lysis of infected cells, were measured. As shown in Fig. 5 (A), the radioactivity was found to be related to the concentration of interferon as indicated by an S-shaped curve which appeared linear in the range corresponding to about 20-75% of radioactivity in the control, or to about 0.25-3 NIH ref. units/ml of interferon. This means that the interferon activity could be reliably detected with 0.1 ml of a sample in the well having more than 0.25 unit/ml of interferon. The concentration of an interferon preparation at which the radioactivity was 50% that in the control was arbitrarily taken as 1 unit/ml, which represented about 0.6 NIH ref. unit/mi. Interferon activity assayed by this method was compared with that assayed by other methods as shown in Fig. 5 (A, B, and C). The activity expressed by this radiochemical microassay method was parallel to, but lower than that expressed by yield reduction of extracellular infectious viruses (A, C). The titer of interferon assayed by this method and that determined by the usual plaque reduction method also seemed to be within the same range (A, B).
Fig.
6.
Time
of
quired for Monolayers plate were ref. units/ml Then
the
contact
in cells
MOI of 20 3H-uridine
the cells
contact with of interferon were
and cultured (1 .0 ,ƒÊCi/ml) counts
were
measured
and
age
of control.
Control
interferon
with in
plotted
VSV
the 24
the
at
culture as the
represented
The
of cold fluids
percent13,056
7.
viruses
an
presence hr.
Fig.
Growth
and
extracellular
1.2, 12 and 120 for various times.
for in
re-
antiviral state. in the microtest
infected
TCA-insoluble
dpm.
with
obtaining of L929
in
L cells
untreated
with
units/ml) cultured
for
Details
at
an
MOI
various
described
left
(10
infected
fluids are
or
interferon
were virus
cell
pretreated
ref. with
of 20
and
in
the
times
presence of 3H-uridine The cold TCA-insolubles culture
of Mengo
interferon-treated
cultures.
Mengo
inhibition
radioactive
(1,ƒÊCi/ml). in the
were
counted. in
the
text.
MICROASSAY
METHOD
FOR
589
INTERFERON
Time of PretreatmentRequiredfor the Cells to Obtain the State of Viral Resistance It is known that the synthesis of the antiviral protein and development of the antiviral state occur rapidly within 2 hr and reach a maximum somewhere between 5 and 8 hr (5) following the contact of cells with interferon. In this radiochemical microassay method, suggestive results were obtained as shown in Fig. 6. At various times of contact of L929 cells with interferon, the cells were infected with VSV, and cold TCA-insolubles in the culture fluid were counted 24 hr after infection. Figure 6 shows that 2 hr contact of the cells with interferon was sufficient in this assay for detecting the relative antiviral activity, and a maximum antiviral state of cells was obtained 8 hr following contact with interferon.
Fig.
8.
Inhibition
dose-response
interferon
of Mengo
viruses
pretreated trations
overnight were infected
with
presence insoluble and
the
of
3H-uridine
counts counts
and
in are
and infecting interferon cultured
(0.51ƒÊCi/ml). the plotted
culture as the
yield-reduction L cells. at
L929
by cells
various concenfor 17 hr in the The
fluids percentage
were
cold
TCA-
measured of control.
Release of Radioactive RNA in L Cell-Mengo Virus System This radiochemical microassay method was employed in another viral infection system and showed a similar tendency. Figure 7 illustrates extracellular RNA released from the infected cells in terms of the incorporation of 3H-uridine into cold TCA-insoluble counts of the culture fluid. Extracellular radioactive RNA was detected as early as 10 hr after infection and continued to increase up to 14 hr. In interferon-treated cells the release of radioactive RNA was clearly inhibited. Figure 8 shows the dose-response inhibition curve in Mengo virus infection obtianed by this assay method. DISCUSSION
Allen and Giron (1) developed an assay method for interferon, in which they assessed the growth of MM virus in terms of the amount of 3H-uridine incorporated
590
H.
into
viral
RNA.
containing the
synthesis
of
containing
this
and
trapped
cellular
cold were
is
rapid
and
in
which
synthesis
in
infected
On
the
other
of
feron
preparations
ously.
The
by
TCA,
cold
Although to
simpler
Assays
and of
with
interferon
virus
may The
existing the
for microtest
yield the
of titers virus
the
same
the
be
when
a
could
be
a
the the
1 hr
yield use
with
extracellular of interferon almost
We thank Drs. M. Iizuka their kind supply of NIH
was
same
to
the
this method radiochemical
inhibition
to
of
viral
in
RNA
with
3H-uridine to
a
which
caused
preincubated
by inter-
simultane-
filter
paper,
were
requires
a
synthesis
fixed
washed
and
shorter
time
than
precedes
the
release
microassay number
of
method samples
within
based the
pancreatic in
3H-labeled
the
compared
radioactivity
RNA
2 to
more
rapid
on
the
inhibition
our of
proposed
are
3 days
a
microassay
of radioactive treatment.
except
assayed
me-
1).
for
however
of
RNAs
buffered
to
here
be
after
assayed.
the
contact
operation,
Mengo
virus.
reduction of RNase
addition
plate,
was
cells,
into
method,
with
with
large
method
infected
as by
added transferred
completed
challenge
microassay the
and
Furthermore, as
as
by
cells
was
radiochemical
efficient
medium
Radioactivity
radioactive
The
method
of samples
used
of
(Fig.
inhibit
with
simplicity.
virus
viral
pads.
microassay
papers
medium to
above-mentioned
expressed
RNA
because
cells,
fiber
have modified cell system, the
in
release
beaker
later
scraped
simplicity (4)
is
simple.
filter
were
the culture
3 hr
The
from
lack
of
radiochemical
the
cells glass
et al VSV-L
to
each
2 to
radiochemical
of
results
from
present of
cultured
for
the
in
far
activity
with
preparation.
principle such same manner
progeny
time
more
well
destruction
here,
one
is
seems
radioactive hundreds
on
infected,
to
ethanol.
Suzuki with a
is very
infected
the
hundreds
it
were later
the
trapped
inhibition
cells,
and
RNA
infection,
present
by
dishes min
replaced
with
still
the
in 30
was
interferon
cells,
infected
obtain
radioactive
after
however,
were
cells
fluid
Although more rapid
expressed
the
at
method
the
were
the
ET AL
added
dehydration
extracellular
dehydrated
the
hours
hand,
was
destruction
the
Six
efficient,
method,
in
and
method. and
RNA
is
RNA
after
assay
activity
L929 D was
TCA-insolubles counted
plaque reduction be more convenient
the
method,
actinomycin
3H-uridine.
saline
thod
In
5 ,ƒÊg/ml
ISHITSUKA
RNase
virus the
as
that
counted.
by
and S. Kobayashi reference interferon.
at
viruses
yield
in Basic
would
Research
of the
of
are
extracellular method
Laboratory,
in the further 10 ƒÊg/ml
digested
data
of
another
be devised cells were
be
Although
by
on
a concentration
reduction
obtained
interferon
based
viruses could the infected
If
progeny
by
method
and not
only shown
3H-labeled mentioned
Toray
Industries,
here. Inc.
REFERENCES
1) 2) 3)
Allen, P.T., and Giron, D.J. 1970. Rapid, sensitive assay for interferons based on the inhibition of MM virus nucleic acid synthesis. Appl. Microbiol. 20: 317. Fantes, K.H. 1966. Purification, concentration and physicochemical properties of interferons, p. 119. In Finter, N.B. (ed), Interferons, North-Holland Publishing Co., Amsterdam. Finter, N.B. 1973. The assay and standardization of interferon and interferon inducers, p. 135. In Finter, N.B. (ed), Interferon and interferon inducers, North-Holland Publishing Co., Amsterdam.
MICROASSAY
4) 5)
METHOD
FOR
INTERFERON
591
Suzuki, J., Akaboshi, T., and Kobayashi, S. 1974. A rapid and simple method for assaying interferon. Japan. J. Microbiol. 18: 449. Wagner, R.R. 1961. Biological studies of interferon. I. Suppression of cellularinfection with Eastern equine encephalomyelitis virus. Virology 13: 323.
Requests for reprints should be addressed to Dr. H. Ishitsuka, Department of Microbiology and Chemotherapy, Nippon Roche Research Center, 200 Kajiwara, Kamakura-city, Kanagawa, Japan.
