Microbiol. Immunol. Vol. 22 (7), 415-426, 1978
A New Microplate Neutralization of Herpes Simplex Aiko
TADA,1
Department
and
of
Kamesaburo
Virology,
Abstract
A
requiring
microplate
of
munized than
1 HSV with
type
HSV
as did
were
selected,
much 1
HSV
reduction
of
equivocal
intermediate
As
chick
embryo
failed
a routine
to
in
the and
which
type in
would
Vero not
(28)
reported
with
that
practical
similar 1
be
cells.
However,
regarded
of
a
1
Science,
use
of
for
student
of Medicine,
a
against
type
of cross
reaction to
type
of
serial
HSV.
strains
No
including
determined
by
with
in
2
Selective
obtained
plaguing
type
immunized
added
stock that
titers
on
herpetic
constant
infections, which
chick
of
the
Vero
a and
embryo
cells
decisive
which
effect
result
of
Therefore, typing
antisera
of in
of Department University
on
the
in
we
desired
the
The
microplate
of Maternal
of Tokyo. 415
fact,
rationale titration
and
the
in
Child
(25).
cell
species
embryo
cells
biological
Yang
et
markers al
did
establish
themselves not
a
for of
identifi-
(HSV-2)
two
chick of
this
coincide
simple
and
improvement
virus.
Health,
al's was
typing
virus
method to
virus for
(CPE)
et
virus
for
simplex
In microplate
Yang
isolated
parallel
herpes
criterion
the
HSV.
in
lower
differentiation. the
test.
2
markedly based
adopted of
antiserum
cells
type
titered typing
of
embryo
cytopathic
we
titration
amount
chick
which
a
the of
with
based
differentiate
serological
type-specific
Post-graduate Faculty
with
serological
method the
as
strain
infectivity.
virus
in
primary
endpoints (HSV-1)
a
typing
for
clearly
strains
were
of
number
im-
of HSV
1 HSV.
of
and
could
on
levels
determine a
sim-
types
pigs
reaction
virus
for were
heterologous
complement
coincided
modifications,
absence
Vero
method
showed
from
with
or
with
The type
procedure
(28)
presence
typing
cells. identify
could
based
the of guinea
cross
step pigs
the
minimal
and
among result
method
of
titration
found
typing
al's
diagnostic
also
(1 :160)
guinea
against Sera
having
antiserum
was
The et
titer
complement-
first
differentiate
level
sera
of the
When
titer.
microplate
either
case
Yang
method
cation
by
the
CRN
same pig
dilution
in
titer
isolates.
sometimes
microplate
high
virus
the
the
as
mostly
homologous
Guinea
estimation
(HSV).
could
sera, the
about
their
virus
of
primary
was
showed
of
fresh
rabbit than
antisera.
and
sera
for
1977)
established
virus
late
29,
test was
simplex the
lower
rabbit
modification
than
2 HSV,
dilutions
many
herpes
hyperimmune
being
10-fold
This
type
with
better
done
of
November
antibody
Science,
Tokyo
neutralization
(CRN)
typing
of Medical
of Tokyo,
publication,•@
serum
neutralizing
plification
for
for Typing
YOSHINO
Institute
University
(Received
Test Virus
Our
School
earlier
of Health
416
A. TADA
AND K.
YOSHINO
experience with the microplate (25) suggested that an antiserum adequately diluted so as to eliminate cross-reacting antibody might serve as such a type-specific serum, because in the microplate neutralization test employing the constant serum-varying virus system only a small amount of antibody sufficed to neutralize an unproportionally great amount of virus, resulting in a marked reduction of the CPE endpoint. Before testing this possibility, our first search was directed to obtain antisera possessing minimal levels of cross-reacting antibody. Rabbits and guinea pigs were immunized and examined individually for complement-requiring (CRN) and ordinary neutralizing (N) antibodies. As a result of this preliminary test, we learned that the guinea pig was a good source for such sera, although individual differences existed. Then, using selected guinea pig antisera, the above-described attempt of typing was performed with a number of stock HSV strains. Results of these tests are reported in this communication. MATERIALS
AND
METHODS
Viruses. HF and UW-268 strains were used as representatives of HSV-1 and HSV-2, respectively. Passage histories of these strains are to be referred to our previous paper (25). For immunization of animals, the viruses were passaged once in primary rabbit kidney cells and culture fluids harvested at time of maximal CPE were stored at -70 C. For in vitroneutralization, the same strains passaged in Vero cells were employed. Other strains used for typing experiments included fresh isolates from dermatological wards of several hospitals tested earlier (25) and those isolated by Dr. T. Kawana in the gynecological ward of the University of Tokyo Hospital. Quantitation of the above representative strains was done by plaque assay in primary chick embryo cells (26) and titers were expressed as plaque-forming units (PFU). Immunizationof animals. Rabbits were injected by the intravenous route with multiple weekly doses of approximately 107 PFU of virus, and each animal was bled one week after the last injection. The schedule of immunization varied, but the bleeding time was consistently later than the 4th week. A more systematic schedule of immunization was set for guinea pigs. Groups of 10 guinea pigs weighing about 300 g were given 2 weekly doses of approximately 106 PFU of either HF or UW-268 strain by intraperitoneal injection and boostered once with a similar dose of virus 7 weeks after the first immunization. Bleeding was done at the end of the 3rd and 8th week. All sera were inactivated by heating at 56 C for 30 min and stored at −20C
before
and
Complement HSV,
as
Hemolytic
tested
Vero
pared
by
medium cell
by
units
the
(MM)
cells
was
sera
by
were
the
the
5•~
105
with
calf with
was
method
grown
above cells
medium per
ml.
of were
nonspecific
and
described
Single
and
stored
earlier minimum
suspensions the
2%
calf
against at -70
C.
(27). essential
cell
suspending containing
inhibitor
pooled
Eagle's
serum.
trypsin
preservatives. free
method,
inactivated
monolayers which
adding
pig
reduction
determined
10%
dispersing
guinea
plaque
Vero with
concentration
tests without
Fresh
were
cells.
supplemented
between
(C).
cells
in
serum.
medium were
pre-
maintenance The
final
MICROPLATE
Microplate plate,
it
MM
plastic
in
tray
serum
wells
MM
incubated Vero
at
cell
plate cells
After to
wells,
In
taken
as
serum,
their
all
the
were
used
were
per
the
as
positive
to
obtain
same
Plaque with
The
A
dilution
of
ml
for
which
taken
as K
values
of
10
20
and
reduction
min, was
wells
of
lines
two
of
was
plate
to
10%
was
the
incu-
formalin
more
than
was
4 hr
grade
0-2
CPE
wells
were
set
positive
lines
also
gave
the
virus
had contained
into
3 dishes
the
were for
a
neutralization
different
case
the
of
endpoints,
when
quadruplicate
by
was
by
of
less
37
in
serum
one
CPW and
drop
was
taken
Muench
(19)
of
per
50%
of
1 hr
an in
Each in
highest
technique
to
egg
mixture
the
amount
serum
control
a
likewise. chicken
ml.
the
dilu-
count
was
reciprocal. The Serum
C
and
C for
normal
The
prepared
treated
monolayer
(26).
than
37
0.05
cell
were dilution
was
emulsion virus
(12).
1 :100 assay
100.5-fold
procedures
serum
at
of
by
been
of
incubated
embryo
at had
1-4 Reed
serum
method
earlier
of
the
neutralization.
diluted plaque
20%
expressed
warmed
strength were
a
to
grade
dilutions
place
chick
stated
saline,
case,
of
PFU
standard
kinetic
that
whose
with
falling
received
Subsequent
formula
and
in
200
in
well
(TCD).
mixed
number
titer
this the
2-fold
diluent
MM
above.
amounts
were
our
plaque and
aliquots examined
Serial Equal
primary
by
followed
virus,
dose
made
with Each
as
In
infective
about
assay
cells
by
the
been
and
of
test.
virus
using
diluted
dropwise.
drop
test.
seed
was
calculated
culture
of
buffered
seed
one
was
determination•@
phosphate
amount
Virus
saline.
endpoint,
value
returned
inducing
was
MM
the
complete or partial absence of 4 (no cells) to 0 (intact mono-
duplicate
the
was
above-described
and for
CPE,
dilution
dilution
neutralization
control
reduced the
4
This
per
neutralization
plaque
essentially
with
When
and
tissue
dilution
inoculated
tion
MM
buffered
bath.
0.05
titer.
8 wells
the
reduction
chorioallantois was
serum
was
violet
from
grade
highest
plate
as
with
The
temperature
appeared graded
calculated.
endpoint
50%
appropriate
of
in
the
phosphate
water
to of
as
and
room
showed
in the microplate.
drops
the
CPE was
the gentian
of C
serum.
given
two
at
Usually
the
was
of virus
and
virus,
of
mean
Titration dilutions,
wells
neutralization
geometric
lines
left
and
0.2%
the
dropping.
When
1 min,
CO2-air.
diluted
disposable
a concentration
diluted for
first
by
dropped.
vibrator
transfer
received
well
to
the
A
II) per
MM
was
5%
was 1 :5.
ml
with
a
of
wells of
neutralization.
reciprocal
flow
all
being
control
positive
expressed
to
with tap water. by the stain, and
case
and
with
Serum
appropriately
on
use
MicroTest
stated, Virus
not
from
0.025
C diluted
C.
consisting
after
dilution.
(Falcon
shaking
dropped
a stain and
was washed as visualized
of
417
did
starting
otherwise
incubator
was
of
We
delivered
Then
After
HERPESVIRUS
system.
culture which
in place
an
4 days,
all
layer).
of
C in
cell
unless
wells.
suspension
bator. added
the
37
ml,
dropped
to
for
alone.
0.1
dilution
increments,
a pipette
MM
per
was
dropped
of
OF
error
2-fold
wells
aid
received unit
omitted, then
the
serum
standard
with
96
by
the
the
tubes
carrying
hemolytic
by
amplified
test
dilutions
Control 1
neutralization
because
with
NEUTRALIZATION
and
was
quickly
adjusted with primary
diluted
to cold chick
mixed 106
PFU
diluent. embryo
estimate
K
appropriately with per The cells,
an ml.
equal After
infectivity and
the
418
A. TADA ANDK. YOSHINO
reduction per min expressed by natural logarithm averaged from the 10-min and 20-min samples was multiplied by the serum dilution factor to give the K value. RESULTS
Basical Analysesof the Microplate Neutralization Test Initially, it was desired to know the sensitivity as well as the type differentiating ability of the microplate neutralization test, in comparison with those of the two standard neutralization tests, plaque reduction and K value determination. An anti-HF (HSV-1) and an anti-UW-268 (HSV-2) hyperimmune rabbit sera were selected and tested against the homologous and heterologous viruses. Each virus was diluted with 0.5-log increments and each dilution was tested with the sera in quadruplicate. The result is set out in Fig. 1. Both the sera exhibited considerably high titers especially where relatively small amounts of virus were used, and the titers against the heterologous viruses were about 8 to 16-fold lower. An interesting observation was that the use of increasingly higher strengths of virus did not proportionately lower the titers obtained. Rather, a 10fold increase in the virus amount used resulted in only approximately 3-fold decrease of the endpoint. The same sera were subjected to plaque reduction and kinetic curve neutralization tests. The results are summarized in Table 1. As a whole, one can notice no marked advantage in any of these three methods as regards the capacity of type
Fig. 1. Dose responses between the virus amount and the serum neutralization endpoint obtained in the microplate without addition of C. Filled circles: anti-HF (HSV-1) rabbit serum. Open circles: anti-UW-268 (HSV-2) rabbit serum. Solid lines: titers against the homologous strain. Broken lines: titers against the heterologous strain.
MICROPLATENEUTRALIZATIONOF HERPESVIRUS
419
differentiation. Hitherto, the kinetic curve test has been considered to be suited for type differentiation. In fact, the anti-HF serum showed an about 16-fold higher value with the homologous than with the heterologous virus. However, the difference was 6-fold in the case of the anti-UW-268 serum. The sensitivity of the microplate method was about the same as that of the plaque reduction test. We then desired to apply the microplate neutralization test for estimation of complement-requiring neutralizing (CRN) antibody. An unexpected finding obtained in a preliminary experiment was that C exerted an increased effect-of nonspecific inhibition in this system due to its constant presence without dilution. Table 2 demonstrates two examples of such tests, in which varying dilutions of fresh guinea pig sera were added to virus dilution series. One of these sera was especially detrimental to UW-268 strain, 102.25TCD being unable to consistently induce.grade 4 CPE in the presence of more than 2 hemolytic units/0.1 ml of C. Of course, a reproducible neutralization endpoint would be expected only when the control wells invariably show grade 4 CPE. Hence, we decided to use 1000 TCD of virus and Table 1. Reciprocal neutralization tests between HF (HSV-1) and UW-268 (HSV-2) strains by the plaque reduction, K value determination and microplate methods
Table
2.
Influence of fresh guinea pig serum upon the CPE induction in the microplate by HSV-1 and HSV-2
420
A. TADA
AND K. YOSHINO
1 hemolytic unit/0.1 ml of C in all cases, at the expense of the 3-fold decrease of endpoint as compared to the test using 100 TCD. The Levelof Cross-ReactingAntibodiesin Rabbit Sera All anti-HF and anti-UW-268 rabbit sera available at hand were tested for homologous and heterologous neutralization titers by the microplate method with or without addition of C. This was done in a hope that some sera would show extremely low levels of cross-reacting antibody, since existence of individual differences of rabbit antisera in the cross reaction has been known (21). As listed in Table 3, the difference between the homologous and heterologous neutralization titers was mostly smaller than 8-fold, and did not materially change even when C was included in the reaction. The difference was averagely smaller in the case of anti-UW-268 sera. Then, 4 rabbits immunized with another HSV-2 strain, Uchiyama, were further tested, but again the difference did not exceed 4-fold. The Levelof Cross-ReactingAntibodiesin GuinesPig Sera Ten guinea pigs were immunized with HF strain and another group of 10 guinea pigs with UW-268 strain. In the latter group two animals died before completion Table
3.
Neutralization the homologous
titers of anti-HSV-1 and anti-HSV-2 rabbit sera against and heterologous strains obtained in the microplate with or without C
MICROPLATE
Fig. 2. against
NEUTRALIZATION
OF
HERPESVIRUS
421
Microplate neutralization tests with anti-HF and anti-UW-268 guinea pig sera the homologous and heterologous strains in the presence or absence of C. The
early and late serum samples were obtained 3 and 8 weeks, respectively, following the first immunization. For each serum sample, the left column denotes titers obtained without C, and the right column those obtained with addition of C. Shadowed portion expresses the titer against the heterologous strain.
of the scheduled immunization and are omitted from the present data. The 3-week (early) and 8-week (late) sera of these animals were tested as above, and the result is presented in Fig. 2. Again, individual differences were noted among the animals receiving the same schedule of immunization. In the case of the anti-HF sera, the difference between the homologous and heterologous titers was similar as in the above rabbit antisera. In contrast, this difference was greater in the case of the late antiUW-268 sera, 5 out of 8 animals revealing differences greater than 8-fold either in the presence or in the absence of C. The late sera of guinea pigs No. 3 and No. 11 were selected as representative anti-HF and anti-UW-268 sera to be used in the following experiments. Effect of a High Dilution of the RepresentativeGuineaPig Antisera upon the Titration of the Homologousand HeterologousVirusesin the Microplate Since the two representative guinea pig antisera both showed an endpoint of 1:320 in the presence of C in the above test in which the indicator virus was 1000 TCD, it was expected that a high dilution of the sera would reduce the TCD titer of the homologous virus as determined in the microplate. Thus, in the next experiment, HF and UW-268 strains were titrated for TCD in a usual manner, except that two lines of wells were used and varying dilutions, from 1 :80 to 1:320, of the
422
A. TADA
AND K. YOSHINO
Fig. 3. Reduction of infectivity titer of HF and UW-268 strains, as estimated in the microplate, due to presence of the homologous or heterologous guinea pig antiserum.
above guinea pig sera and C were added. Controls were set omitting the addition of antisera. TCD titers thus obtained are compared in Fig. 3. As expected, these high dilutions of the antisera selectively reduced the TCD titer of the homologous virus. Some extent of cross reaction of the anti-HF serum was seen, but the cross reaction was negligible in the case of the anti-UW-268 serum. Typing of VariousHSV Strains in the Microplate Each stock virus, in a form of culture fluid of Vero or secondary rabbit kidney cells, was diluted with MM as usual. The above representative guinea pig sera were also diluted with MM both 1:160. Six lines of wells of a microplate were used for titration of the virus, each well receiving one drop of virus dilution, one drop of C, one drop of antiserum and one drop of Vero cells. The first two lines of wells received the anti-HF and next 2 lines the anti-UW-268 serum, while the remaining 2 lines served as control, receiving MM in place of antiserum. Reduction of CPE endpoint by antibody was expressed in loglo. When the reduction by the anti-HF serum was greater than that by the anti-UW-268 serum, the virus was registered as HSV-1, and in a reverse case it was judged to be HSV-2. Results obtained with a number of stock strains are plotted in Fig. 4, which shows that no equivocal intermediate case was present between the two groups of HSV-1 and HSV-2. Comparisonwith Typing by BiologicalMarkers Among various methods for typing of HSV, those commonly practiced may be
MICROPLATE
NEUTRALIZATION
OF HERPES
423
VIRUS
Fig. 4. Differentiation between HSV-1 and HSV2 by the microplate neutralization test. Each strain was titrated in the microplate in the presence of (i) a standard anti-HF guinea pig serum diluted 1:160, (ii) a standard anti-UW-268 guinea pig serum diluted 1 :160, and (iii) MM (control). All strains tested are plotted according to the titer reductions by the two sera.
Table
4.
Comparison
of the typing by other
by the microplate methods
method
with
that
A. TADA
424
Yang
et
chick
embryo
al's
plaques of HSV-1 our
or
microplate
if the isolates.
result
but
of
cells.
The
the
The
plaque All
modification
embryo
method
cells.
the
or
its
modification
determines
an
Yang
et of
al's
method
these
typing
method
(25)
tests
are
perfectly
could
not
and
isolate
size is small, but sometimes the strains described in the
present
plaguing
(28)
latter
Results
of
AND K. YOSHINO
as
in
if it
primary
fails
to
gives a false identification preceding chapter were
and
also
for
combined
plaques
for
coincided
identify
plaguing
HSV-1
with
some
in
HSV-1
of
chick
in
Yang
case by
primary
presentation
that
form
in tested
et
Table
al's
4.
method,
strains.
DISCUSSION
The or
by
type
tests
embryo
cells
obtained of
determination
for
(4,
with
the
14,
a
drawback
HSV-1
and The
(11)
among
too
(16-18,
HSV
or
common
present
of
fit
system
replaced
by
plaque titration a considerable
exceed
using We
has
chick
virus
preferred
titers because
serological
tests. antigenic
serological
cross
the
proved
the
learned
some a
tests,
reaction
a
between
that
consumed
endsome et al
was
type-
was IgM
of or
by
of the
cells
found
so much
infected
the
test.
type-specific
decades the
suffices
titer
decrease
prejudice
virus before,
but
development
serum-varying Also, since
(3),
of
reason
serum-varying one
it is
failure
antibody
after
this
the
with
same
a high-titered
a common
However,
for
type-specific
system
absorption
However,
Also,
constant
constant for one
by
(2).
answer
of
amount
be
sera
HSV
laboratories.
unneutralized
range.
IgM strain
type-specific
a decisive
the
of
virus, and Kawana
rabbit
test
type
serum
case of is required
of virus
lirnited
this
virus
to
isolate types.
intermediate
production
used
measurement an two
needed.
one
give
virus-varying
portion
certain
no
adopting
the
with the
diagnostic
a small
neutralization
constant
only
not
only
employed
hyperimmune
that
was
in
neutralization
of
that
heterologous
would
but
all
to produce
practice
of viruses. In amount of•@antibody
leaves
not
high
means
primary
require
for
the
However,
for by
line
necessary by
(7)
attempted
the
cell
HSV
This
antiserum
be
course
required
sera between
stated
susceptible
method
is not
system.
in
laborious of
been
29)
preparations
antibody
to
and tested.
IgM
cells
in a resistant above.
typing
they
of
20,
et al
C,
of investigation 22)
cumbersome
The
labor has
may
the
should
and means
serological
comparing
(28)
with
and anti-HSV-2 intermediate
with of
lines 8-10,
antiserum
cells
types
by
plaguing method
embryo
time this
either
especially
compared
of Hamper
supply
of
growing as stated
when
the
performed
microplate
of
tests
finding
isolates
absorption
does
as
differentiation
serological
the
Other 5, 6,
al's
chick
accompanying
a constant
was
technique
neutralization
the
that
et
and
from
been latter,
HSV-2.
utilized
for
Yang
cells
of
earlier
in
has The
of standard anti-HSV-1 were recorded as being
specific
(1,
and
Aside
definite
points strains
15)
decisive
determination.
HSV
markers.
susceptible
simplicity
However,
of
biological
virus even by
should
of system, a potent
antiserum be revised
microplate. this
while
performing
the
previous
study
on
the
isolation
of HSV
MICROPLATE
(25).
In
that
presence
or
tion. in
study,
CPE
absence
of
Amazingly, the
We
same
The
Thus,
success
in
to
HSV-2
clear
more
pigs
the
the
microplate
in
purpose
well
of
sometimes
attempt
titered
present from C
the
virus
in
after
also
be
this
function
as
cells
may
zero
from
system
released
the
identifica-
stemmed
initially
of
in
some
other
multiple
ascribable
Whatever
to
proves
of of
obviating
itself
handle a
serum
a
can
serum
of
which
thousands
This
work
be
of
aided
by
antisera
pigs
guinea Sephadex
G-200
fraction. in
case
and
their
In
the
this
of
We
that the
level
15
were
No. CRN
antibody
connection,
guinea
do
thought
above
11
in anti-
24).
we
antibody No.
pig was
that
21,
first,
be
guinea This
(13,
At IgM
IgG
late
indicating
sera
of
that
the
could contributing
antibody.
type-specific
the
phenomenon pig
may
antisera
practical
may be
pigs
would
late
a grant
from
the
the
we
are
IgG
re-
with antibody
enabled
of
Ministry
ease. the
as
present
and
in
pigs
are
Secondly, in
1 :160
the
of Health
guinea
animals
used, a
present
high
system,
Furthermore,
of by
high neutrali-
because
procedures. dilution
averagely
microplate
number
type-specific
a
be
large
accomplished
at
valid,
present First,
a
absorption
used
be the
importance.
as
or
in
when
be
used
be
strains
that
anti-HSV-1
especially
can
of
was
handicaped
guinea
fractionation
available
fact
phenomenon.
(23)
the
of
rabbits
standard
such laborious
are
to
than
method
Another
properties.
for
ability
to
selection
in
explanation
rabbit
through
report
present
was
with
sera
physicochemical
type-differentiating test
much
filtration
al's
types cross-reacting
this
late
the
of
than
predominantly et
HSV
level
have
by antisera.
workers
the
gel
typing
of low
for
when
Shinkai
IgM
dilution
to
but
of
easier
for unabsorbed
other
happened
resided
sembles
on
the virus
of
explanation
by
reminded
typing
per
present
inhibition
cross-reactive
plausible
antibody,
activity
sera
with
utilizable
a
of
chromatographed
are
in
neutralization
upon
differentiation showed
were
any
guinea
zation
105
microplate
nonspecific
dilution
experiences
sera have
IgG
mere
HSV-2
contrast
of
the
done for
the
reaction
sera
by
against
the
than
continues
increased
type-specific easily
the
not
in
after
but
was antiserum
underlying
425
HERPESVIRUS
reason.
provided
sera
end
isolates
higher
idea that
OF
rabbit
titering
systems, cycles.
the
to
not
of
diluted
The
consider
does
neutralization growth
1 :20.
series.
antibody
titration
a
strains
antiserum
observation. of
NEUTRALIZATION
if
the
anti-
present
data,
method.
Welfare
of Japan
for
research
children.
REFERENCES
1)
Back,
A.F.,
and
inhibition 2)
Docherty, viruses Gen.
3)
J. J.,
Virol.
Schmidt,
R., animal
Figueroa,
Forghani,
and
genital B.,
Typing
and
Rapp,
lines
established
and
Strickland,
Herpesvirus reaction.
F. after
hominis
Appl.
antibodies
Microbiol.
1971.
Differential
exposure
to
28:
and
susceptibility
chemically
isolates
by
400-405. to
inactivated
herpes
simplex
herpes
virus.
377-384.
Vogt,
Schmidt,
cation•@ of Herpesvirus
1974.
hemagglutination F. J.,
cell
viruses,
M.E., and
N.J.
indirect O'Neill,
13:
Dulbecco,
of oral 5)
the
of hamster
of two 4)
of
M.,
western
equine
Rawls, origin. N.J., hominis
W.E. J.
Gen. and types
A.G.R.
1956.
encephalitis 1969. Virol.
virus
Biological 4:
A study and
markers
of the
basic
poliomyelitis
aspects virus.
of differentiation
of neutralization
Virology
2:
of herpes-virus
162-205. strains
259-267.
Lennette,
E.H.
1974.
1 and
2 from
clinical
Solid
phase
materials.
radioimmunoassay Appl.
Microbiol.
for 28:
identifi661-667.
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6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23) 24)
25) 26) 27) 28) 29)
A. TADA
AND K. YOSHINO
Geder, L., and Skinner, G.R.B. 1971. Differentiation between type 1 and 2 strains of herpes simplex virus by an indirect immunofluorescent technique. J. Gen. Virol. 12: 179-182. Hampar, B., Martos, L.M., Chakrabarty, M., and Burroughs, M.A.K. 1970. Late 19s rabbit antibody neutralization test for differentiating herpes simplex virus types 1 and 2. J. Immunol. 104: 593-598. Ito, M., and Barron, A.L. 1974. Typing of isolates of herpes simplex virus by mixed agglutination. Proc. Soc. Exp. Biol. Med. 146: 41-45. Jeansson, S. 1972. Differentiation between herpes simplex virus type 1 and 2 strains by immunoelectroosmophoresis. Appl. Microbiol. 24: 96-100. Jeansson, S. 1975. Preparation of type specific herpes simplex antisera by an immunosorbent method. Acta Pathol. Microbiol. Scand. Sect. B 83: 48-54. Kawana, T., Shinkai, K., and Yoshino, K. 1974. Typing of herpes simplex virus strains of genital and nongenital origins. Japan. J. Microbiol. 18: 235-241. Kawana, T., Yoshino, K., and Kasamatsu, T. 1974. Estimation of specific antibody to type 2 herpes simplex virus among patients with carcinoma of the uterine cervix. Gann (Tokyo) 65: 439-445. Leinikki, P. 1971. Immunofluorescent assay of herpesvirus type 1 and 2 antibodies in rabbit and human sera. Arch. Ges. Virusforsch. 35: 349-355. Lowry, S.P., Melnick, J.L., and Rawls, W.E. 1971. Investigation of plaque formation in chick embryo cells as a biological marker for distinguishing herpes virus type 2 from type 1. J. Gen. Virol. 10: 1-9. Marks-Hellman, S., and Ho, M. 1976. Use of biological characteristics to type Herpesvirus hominis types 1 and 2 in diagnostic laboratories. J. Clin. Microbiol. 3: 277-280. Nahmias, A.J., Chiang, W.T., delBuono, I., and Duffey, A. 1969. Typing of Herpesvirus hominis strains by a direct immunofluorescent technique. Proc. Soc. Exp. Biol. Med. 132: 386-390. Nahmias, A.J., Dowdle, W.R., Kramer, J.H., Luce, C.F., and Mansour, S.C. 1969. Antibodies to Herpesvirus hominis types 1 and 2 in the rabbit. J.Immunol. 102: 956-962. Pauls, F.P., and Dowdle, W.R. 1967. A serologic study of Herpesvirus hominis strains by microneutralization tests. J. Immunol. 98: 941-947. Reed, L., and Muench, H. 1938. A simple method estimating fifty per cent endpoints. Am. J. Hyg. 27: 493-497. Roizman, B., Keller, J.M., Spear, P.G., Terni, M., Nahmias, A.J., and Dowdle, W.R. 1970. Variability, structural glycoproteins, and classification of herpes simplex virus. Nature 227: 1253-1254. Savage, T., Roizman, B., and Heine, J.W. 1972. Immunological specificity of the glycoproteins of herpes simplex virus subtypes 1 and 2. J. Gen. Virol. 17: 31-48. Schneweis, K.E., and Nahmias, A.J. 1971. Antigens of herpes simplex virus type 1 and 2 -immunodiffusion and inhibition passive hemagglutination studies. Z. Immun.-Forsch. 141: 471-487. Shinkai, K., and Yoshino, K. 1975. Complement requirement of neutralizing antibodies in different classes of immunoglobulin appearing in rabbits and guinea pigs after primary and booster immunizations with herpes simplex virus. Japan. J. Microbiol. 19: 25-34. Skinner, G.R.B., Thouless, M.E., Trueman, S., Edwards, J., and Gibbs, A.J. 1976. Serological relatedness of herpes simplex viruses. Type-specificity of antibody response. Immunology 31: 481-494 . Tada, A., Sekine, N., Toba, M., and Yoshino, K.1977. An analysis of factors influencing the isolation rate of herpes simplex virus. Microbiol. Immunol. 21: 219-229. Taniguchi, S., and Yoshino, K. 1964. An analysis of the plaque assay of herpes simplex virus in chick embryo monolayers. Arch. Ges. Virusforsch. 14: 537-552. Taniguchi, S., and Yoshino, K. 1965. Studies on the neutralization of herpes simplex virus. II. Analysis of complement as the antibody-potentiating factor. Virology 26: 54-60. Yang, J.S.P., Chiang, W., Gale, J.L., and Chen, N.S.T. 1975. A chick-embryo cell microtest for typing of Herpesvirus hominis. Proc. Soc. Exp. Biol. Med. 148: 324-328. Wheeler, C.E., Jr., Briggaman, R.A., and Henderson, R.R. 1969. Discrimination between two strains (types) of herpes simplex virus by various modifications of the neutralization test. J. Immunol. 102: 1179-1192.
Requests for reprints should be addressed to Dr. Aiko Tada, Department of Maternal and child Health, School of Health Science, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
A New Microplate Neutralization of Herpes Simplex Aiko
TADA,1
Department
and
of
Kamesaburo
Virology,
Abstract
A
requiring
microplate
of
munized than
1 HSV with
type
HSV
as did
were
selected,
much 1
HSV
reduction
of
equivocal
intermediate
As
chick
embryo
failed
a routine
to
in
the and
which
type in
would
Vero not
(28)
reported
with
that
practical
similar 1
be
cells.
However,
regarded
of
a
1
Science,
use
of
for
student
of Medicine,
a
against
type
of cross
reaction to
type
of
serial
HSV.
strains
No
including
determined
by
with
in
2
Selective
obtained
plaguing
type
immunized
added
stock that
titers
on
herpetic
constant
infections, which
chick
of
the
Vero
a and
embryo
cells
decisive
which
effect
result
of
Therefore, typing
antisera
of in
of Department University
on
the
in
we
desired
the
The
microplate
of Maternal
of Tokyo. 415
fact,
rationale titration
and
the
in
Child
(25).
cell
species
embryo
cells
biological
Yang
et
markers al
did
establish
themselves not
a
for of
identifi-
(HSV-2)
two
chick of
this
coincide
simple
and
improvement
virus.
Health,
al's was
typing
virus
method to
virus for
(CPE)
et
virus
for
simplex
In microplate
Yang
isolated
parallel
herpes
criterion
the
HSV.
in
lower
differentiation. the
test.
2
markedly based
adopted of
antiserum
cells
type
titered typing
of
embryo
cytopathic
we
titration
amount
chick
which
a
the of
with
based
differentiate
serological
type-specific
Post-graduate Faculty
with
serological
method the
as
strain
infectivity.
virus
in
primary
endpoints (HSV-1)
a
typing
for
clearly
strains
were
of
number
im-
of HSV
1 HSV.
of
and
could
on
levels
determine a
sim-
types
pigs
reaction
virus
for were
heterologous
complement
coincided
modifications,
absence
Vero
method
showed
from
with
or
with
The type
procedure
(28)
presence
typing
cells. identify
could
based
the of guinea
cross
step pigs
the
minimal
and
among result
method
of
titration
found
typing
al's
diagnostic
also
(1 :160)
guinea
against Sera
having
antiserum
was
The et
titer
complement-
first
differentiate
level
sera
of the
When
titer.
microplate
either
case
Yang
method
cation
by
the
CRN
same pig
dilution
in
titer
isolates.
sometimes
microplate
high
virus
the
the
as
mostly
homologous
Guinea
estimation
(HSV).
could
sera, the
about
their
virus
of
primary
was
showed
of
fresh
rabbit than
antisera.
and
sera
for
1977)
established
virus
late
29,
test was
simplex the
lower
rabbit
modification
than
2 HSV,
dilutions
many
herpes
hyperimmune
being
10-fold
This
type
with
better
done
of
November
antibody
Science,
Tokyo
neutralization
(CRN)
typing
of Medical
of Tokyo,
publication,•@
serum
neutralizing
plification
for
for Typing
YOSHINO
Institute
University
(Received
Test Virus
Our
School
earlier
of Health
416
A. TADA
AND K.
YOSHINO
experience with the microplate (25) suggested that an antiserum adequately diluted so as to eliminate cross-reacting antibody might serve as such a type-specific serum, because in the microplate neutralization test employing the constant serum-varying virus system only a small amount of antibody sufficed to neutralize an unproportionally great amount of virus, resulting in a marked reduction of the CPE endpoint. Before testing this possibility, our first search was directed to obtain antisera possessing minimal levels of cross-reacting antibody. Rabbits and guinea pigs were immunized and examined individually for complement-requiring (CRN) and ordinary neutralizing (N) antibodies. As a result of this preliminary test, we learned that the guinea pig was a good source for such sera, although individual differences existed. Then, using selected guinea pig antisera, the above-described attempt of typing was performed with a number of stock HSV strains. Results of these tests are reported in this communication. MATERIALS
AND
METHODS
Viruses. HF and UW-268 strains were used as representatives of HSV-1 and HSV-2, respectively. Passage histories of these strains are to be referred to our previous paper (25). For immunization of animals, the viruses were passaged once in primary rabbit kidney cells and culture fluids harvested at time of maximal CPE were stored at -70 C. For in vitroneutralization, the same strains passaged in Vero cells were employed. Other strains used for typing experiments included fresh isolates from dermatological wards of several hospitals tested earlier (25) and those isolated by Dr. T. Kawana in the gynecological ward of the University of Tokyo Hospital. Quantitation of the above representative strains was done by plaque assay in primary chick embryo cells (26) and titers were expressed as plaque-forming units (PFU). Immunizationof animals. Rabbits were injected by the intravenous route with multiple weekly doses of approximately 107 PFU of virus, and each animal was bled one week after the last injection. The schedule of immunization varied, but the bleeding time was consistently later than the 4th week. A more systematic schedule of immunization was set for guinea pigs. Groups of 10 guinea pigs weighing about 300 g were given 2 weekly doses of approximately 106 PFU of either HF or UW-268 strain by intraperitoneal injection and boostered once with a similar dose of virus 7 weeks after the first immunization. Bleeding was done at the end of the 3rd and 8th week. All sera were inactivated by heating at 56 C for 30 min and stored at −20C
before
and
Complement HSV,
as
Hemolytic
tested
Vero
pared
by
medium cell
by
units
the
(MM)
cells
was
sera
by
were
the
the
5•~
105
with
calf with
was
method
grown
above cells
medium per
ml.
of were
nonspecific
and
described
Single
and
stored
earlier minimum
suspensions the
2%
calf
against at -70
C.
(27). essential
cell
suspending containing
inhibitor
pooled
Eagle's
serum.
trypsin
preservatives. free
method,
inactivated
monolayers which
adding
pig
reduction
determined
10%
dispersing
guinea
plaque
Vero with
concentration
tests without
Fresh
were
cells.
supplemented
between
(C).
cells
in
serum.
medium were
pre-
maintenance The
final
MICROPLATE
Microplate plate,
it
MM
plastic
in
tray
serum
wells
MM
incubated Vero
at
cell
plate cells
After to
wells,
In
taken
as
serum,
their
all
the
were
used
were
per
the
as
positive
to
obtain
same
Plaque with
The
A
dilution
of
ml
for
which
taken
as K
values
of
10
20
and
reduction
min, was
wells
of
lines
two
of
was
plate
to
10%
was
the
incu-
formalin
more
than
was
4 hr
grade
0-2
CPE
wells
were
set
positive
lines
also
gave
the
virus
had contained
into
3 dishes
the
were for
a
neutralization
different
case
the
of
endpoints,
when
quadruplicate
by
was
by
of
less
37
in
serum
one
CPW and
drop
was
taken
Muench
(19)
of
per
50%
of
1 hr
an in
Each in
highest
technique
to
egg
mixture
the
amount
serum
control
a
likewise. chicken
ml.
the
dilu-
count
was
reciprocal. The Serum
C
and
C for
normal
The
prepared
treated
monolayer
(26).
than
37
0.05
cell
were dilution
was
emulsion virus
(12).
1 :100 assay
100.5-fold
procedures
serum
at
of
by
been
of
incubated
embryo
at had
1-4 Reed
serum
method
earlier
of
the
neutralization.
diluted plaque
20%
expressed
warmed
strength were
a
to
grade
dilutions
place
chick
stated
saline,
case,
of
PFU
standard
kinetic
that
whose
with
falling
received
Subsequent
formula
and
in
200
in
well
(TCD).
mixed
number
titer
this the
2-fold
diluent
MM
above.
amounts
were
our
plaque and
aliquots examined
Serial Equal
primary
by
followed
virus,
dose
made
with Each
as
In
infective
about
assay
cells
by
the
been
and
of
test.
virus
using
diluted
dropwise.
drop
test.
seed
was
calculated
culture
of
buffered
seed
one
was
determination•@
phosphate
amount
Virus
saline.
endpoint,
value
returned
inducing
was
MM
the
complete or partial absence of 4 (no cells) to 0 (intact mono-
duplicate
the
was
above-described
and for
CPE,
dilution
dilution
neutralization
control
reduced the
4
This
per
neutralization
plaque
essentially
with
When
and
tissue
dilution
inoculated
tion
MM
buffered
bath.
0.05
titer.
8 wells
the
reduction
chorioallantois was
serum
was
violet
from
grade
highest
plate
as
with
The
temperature
appeared graded
calculated.
endpoint
50%
appropriate
of
in
the
phosphate
water
to of
as
and
room
showed
in the microplate.
drops
the
CPE was
the gentian
of C
serum.
given
two
at
Usually
the
was
of virus
and
virus,
of
mean
Titration dilutions,
wells
neutralization
geometric
lines
left
and
0.2%
the
dropping.
When
1 min,
CO2-air.
diluted
disposable
a concentration
diluted for
first
by
dropped.
vibrator
transfer
received
well
to
the
A
II) per
MM
was
5%
was 1 :5.
ml
with
a
of
wells of
neutralization.
reciprocal
flow
all
being
control
positive
expressed
to
with tap water. by the stain, and
case
and
with
Serum
appropriately
on
use
MicroTest
stated, Virus
not
from
0.025
C diluted
C.
consisting
after
dilution.
(Falcon
shaking
dropped
a stain and
was washed as visualized
of
417
did
starting
otherwise
incubator
was
of
We
delivered
Then
After
HERPESVIRUS
system.
culture which
in place
an
4 days,
all
layer).
of
C in
cell
unless
wells.
suspension
bator. added
the
37
ml,
dropped
to
for
alone.
0.1
dilution
increments,
a pipette
MM
per
was
dropped
of
OF
error
2-fold
wells
aid
received unit
omitted, then
the
serum
standard
with
96
by
the
the
tubes
carrying
hemolytic
by
amplified
test
dilutions
Control 1
neutralization
because
with
NEUTRALIZATION
and
was
quickly
adjusted with primary
diluted
to cold chick
mixed 106
PFU
diluent. embryo
estimate
K
appropriately with per The cells,
an ml.
equal After
infectivity and
the
418
A. TADA ANDK. YOSHINO
reduction per min expressed by natural logarithm averaged from the 10-min and 20-min samples was multiplied by the serum dilution factor to give the K value. RESULTS
Basical Analysesof the Microplate Neutralization Test Initially, it was desired to know the sensitivity as well as the type differentiating ability of the microplate neutralization test, in comparison with those of the two standard neutralization tests, plaque reduction and K value determination. An anti-HF (HSV-1) and an anti-UW-268 (HSV-2) hyperimmune rabbit sera were selected and tested against the homologous and heterologous viruses. Each virus was diluted with 0.5-log increments and each dilution was tested with the sera in quadruplicate. The result is set out in Fig. 1. Both the sera exhibited considerably high titers especially where relatively small amounts of virus were used, and the titers against the heterologous viruses were about 8 to 16-fold lower. An interesting observation was that the use of increasingly higher strengths of virus did not proportionately lower the titers obtained. Rather, a 10fold increase in the virus amount used resulted in only approximately 3-fold decrease of the endpoint. The same sera were subjected to plaque reduction and kinetic curve neutralization tests. The results are summarized in Table 1. As a whole, one can notice no marked advantage in any of these three methods as regards the capacity of type
Fig. 1. Dose responses between the virus amount and the serum neutralization endpoint obtained in the microplate without addition of C. Filled circles: anti-HF (HSV-1) rabbit serum. Open circles: anti-UW-268 (HSV-2) rabbit serum. Solid lines: titers against the homologous strain. Broken lines: titers against the heterologous strain.
MICROPLATENEUTRALIZATIONOF HERPESVIRUS
419
differentiation. Hitherto, the kinetic curve test has been considered to be suited for type differentiation. In fact, the anti-HF serum showed an about 16-fold higher value with the homologous than with the heterologous virus. However, the difference was 6-fold in the case of the anti-UW-268 serum. The sensitivity of the microplate method was about the same as that of the plaque reduction test. We then desired to apply the microplate neutralization test for estimation of complement-requiring neutralizing (CRN) antibody. An unexpected finding obtained in a preliminary experiment was that C exerted an increased effect-of nonspecific inhibition in this system due to its constant presence without dilution. Table 2 demonstrates two examples of such tests, in which varying dilutions of fresh guinea pig sera were added to virus dilution series. One of these sera was especially detrimental to UW-268 strain, 102.25TCD being unable to consistently induce.grade 4 CPE in the presence of more than 2 hemolytic units/0.1 ml of C. Of course, a reproducible neutralization endpoint would be expected only when the control wells invariably show grade 4 CPE. Hence, we decided to use 1000 TCD of virus and Table 1. Reciprocal neutralization tests between HF (HSV-1) and UW-268 (HSV-2) strains by the plaque reduction, K value determination and microplate methods
Table
2.
Influence of fresh guinea pig serum upon the CPE induction in the microplate by HSV-1 and HSV-2
420
A. TADA
AND K. YOSHINO
1 hemolytic unit/0.1 ml of C in all cases, at the expense of the 3-fold decrease of endpoint as compared to the test using 100 TCD. The Levelof Cross-ReactingAntibodiesin Rabbit Sera All anti-HF and anti-UW-268 rabbit sera available at hand were tested for homologous and heterologous neutralization titers by the microplate method with or without addition of C. This was done in a hope that some sera would show extremely low levels of cross-reacting antibody, since existence of individual differences of rabbit antisera in the cross reaction has been known (21). As listed in Table 3, the difference between the homologous and heterologous neutralization titers was mostly smaller than 8-fold, and did not materially change even when C was included in the reaction. The difference was averagely smaller in the case of anti-UW-268 sera. Then, 4 rabbits immunized with another HSV-2 strain, Uchiyama, were further tested, but again the difference did not exceed 4-fold. The Levelof Cross-ReactingAntibodiesin GuinesPig Sera Ten guinea pigs were immunized with HF strain and another group of 10 guinea pigs with UW-268 strain. In the latter group two animals died before completion Table
3.
Neutralization the homologous
titers of anti-HSV-1 and anti-HSV-2 rabbit sera against and heterologous strains obtained in the microplate with or without C
MICROPLATE
Fig. 2. against
NEUTRALIZATION
OF
HERPESVIRUS
421
Microplate neutralization tests with anti-HF and anti-UW-268 guinea pig sera the homologous and heterologous strains in the presence or absence of C. The
early and late serum samples were obtained 3 and 8 weeks, respectively, following the first immunization. For each serum sample, the left column denotes titers obtained without C, and the right column those obtained with addition of C. Shadowed portion expresses the titer against the heterologous strain.
of the scheduled immunization and are omitted from the present data. The 3-week (early) and 8-week (late) sera of these animals were tested as above, and the result is presented in Fig. 2. Again, individual differences were noted among the animals receiving the same schedule of immunization. In the case of the anti-HF sera, the difference between the homologous and heterologous titers was similar as in the above rabbit antisera. In contrast, this difference was greater in the case of the late antiUW-268 sera, 5 out of 8 animals revealing differences greater than 8-fold either in the presence or in the absence of C. The late sera of guinea pigs No. 3 and No. 11 were selected as representative anti-HF and anti-UW-268 sera to be used in the following experiments. Effect of a High Dilution of the RepresentativeGuineaPig Antisera upon the Titration of the Homologousand HeterologousVirusesin the Microplate Since the two representative guinea pig antisera both showed an endpoint of 1:320 in the presence of C in the above test in which the indicator virus was 1000 TCD, it was expected that a high dilution of the sera would reduce the TCD titer of the homologous virus as determined in the microplate. Thus, in the next experiment, HF and UW-268 strains were titrated for TCD in a usual manner, except that two lines of wells were used and varying dilutions, from 1 :80 to 1:320, of the
422
A. TADA
AND K. YOSHINO
Fig. 3. Reduction of infectivity titer of HF and UW-268 strains, as estimated in the microplate, due to presence of the homologous or heterologous guinea pig antiserum.
above guinea pig sera and C were added. Controls were set omitting the addition of antisera. TCD titers thus obtained are compared in Fig. 3. As expected, these high dilutions of the antisera selectively reduced the TCD titer of the homologous virus. Some extent of cross reaction of the anti-HF serum was seen, but the cross reaction was negligible in the case of the anti-UW-268 serum. Typing of VariousHSV Strains in the Microplate Each stock virus, in a form of culture fluid of Vero or secondary rabbit kidney cells, was diluted with MM as usual. The above representative guinea pig sera were also diluted with MM both 1:160. Six lines of wells of a microplate were used for titration of the virus, each well receiving one drop of virus dilution, one drop of C, one drop of antiserum and one drop of Vero cells. The first two lines of wells received the anti-HF and next 2 lines the anti-UW-268 serum, while the remaining 2 lines served as control, receiving MM in place of antiserum. Reduction of CPE endpoint by antibody was expressed in loglo. When the reduction by the anti-HF serum was greater than that by the anti-UW-268 serum, the virus was registered as HSV-1, and in a reverse case it was judged to be HSV-2. Results obtained with a number of stock strains are plotted in Fig. 4, which shows that no equivocal intermediate case was present between the two groups of HSV-1 and HSV-2. Comparisonwith Typing by BiologicalMarkers Among various methods for typing of HSV, those commonly practiced may be
MICROPLATE
NEUTRALIZATION
OF HERPES
423
VIRUS
Fig. 4. Differentiation between HSV-1 and HSV2 by the microplate neutralization test. Each strain was titrated in the microplate in the presence of (i) a standard anti-HF guinea pig serum diluted 1:160, (ii) a standard anti-UW-268 guinea pig serum diluted 1 :160, and (iii) MM (control). All strains tested are plotted according to the titer reductions by the two sera.
Table
4.
Comparison
of the typing by other
by the microplate methods
method
with
that
A. TADA
424
Yang
et
chick
embryo
al's
plaques of HSV-1 our
or
microplate
if the isolates.
result
but
of
cells.
The
the
The
plaque All
modification
embryo
method
cells.
the
or
its
modification
determines
an
Yang
et of
al's
method
these
typing
method
(25)
tests
are
perfectly
could
not
and
isolate
size is small, but sometimes the strains described in the
present
plaguing
(28)
latter
Results
of
AND K. YOSHINO
as
in
if it
primary
fails
to
gives a false identification preceding chapter were
and
also
for
combined
plaques
for
coincided
identify
plaguing
HSV-1
with
some
in
HSV-1
of
chick
in
Yang
case by
primary
presentation
that
form
in tested
et
Table
al's
4.
method,
strains.
DISCUSSION
The or
by
type
tests
embryo
cells
obtained of
determination
for
(4,
with
the
14,
a
drawback
HSV-1
and The
(11)
among
too
(16-18,
HSV
or
common
present
of
fit
system
replaced
by
plaque titration a considerable
exceed
using We
has
chick
virus
preferred
titers because
serological
tests. antigenic
serological
cross
the
proved
the
learned
some a
tests,
reaction
a
between
that
consumed
endsome et al
was
type-
was IgM
of or
by
of the
cells
found
so much
infected
the
test.
type-specific
decades the
suffices
titer
decrease
prejudice
virus before,
but
development
serum-varying Also, since
(3),
of
reason
serum-varying one
it is
failure
antibody
after
this
the
with
same
a high-titered
a common
However,
for
type-specific
system
absorption
However,
Also,
constant
constant for one
by
(2).
answer
of
amount
be
sera
HSV
laboratories.
unneutralized
range.
IgM strain
type-specific
a decisive
the
of
virus, and Kawana
rabbit
test
type
serum
case of is required
of virus
lirnited
this
virus
to
isolate types.
intermediate
production
used
measurement an two
needed.
one
give
virus-varying
portion
certain
no
adopting
the
with the
diagnostic
a small
neutralization
constant
only
not
only
employed
hyperimmune
that
was
in
neutralization
of
that
heterologous
would
but
all
to produce
practice
of viruses. In amount of•@antibody
leaves
not
high
means
primary
require
for
the
However,
for by
line
necessary by
(7)
attempted
the
cell
HSV
This
antiserum
be
course
required
sera between
stated
susceptible
method
is not
system.
in
laborious of
been
29)
preparations
antibody
to
and tested.
IgM
cells
in a resistant above.
typing
they
of
20,
et al
C,
of investigation 22)
cumbersome
The
labor has
may
the
should
and means
serological
comparing
(28)
with
and anti-HSV-2 intermediate
with of
lines 8-10,
antiserum
cells
types
by
plaguing method
embryo
time this
either
especially
compared
of Hamper
supply
of
growing as stated
when
the
performed
microplate
of
tests
finding
isolates
absorption
does
as
differentiation
serological
the
Other 5, 6,
al's
chick
accompanying
a constant
was
technique
neutralization
the
that
et
and
from
been latter,
HSV-2.
utilized
for
Yang
cells
of
earlier
in
has The
of standard anti-HSV-1 were recorded as being
specific
(1,
and
Aside
definite
points strains
15)
decisive
determination.
HSV
markers.
susceptible
simplicity
However,
of
biological
virus even by
should
of system, a potent
antiserum be revised
microplate. this
while
performing
the
previous
study
on
the
isolation
of HSV
MICROPLATE
(25).
In
that
presence
or
tion. in
study,
CPE
absence
of
Amazingly, the
We
same
The
Thus,
success
in
to
HSV-2
clear
more
pigs
the
the
microplate
in
purpose
well
of
sometimes
attempt
titered
present from C
the
virus
in
after
also
be
this
function
as
cells
may
zero
from
system
released
the
identifica-
stemmed
initially
of
in
some
other
multiple
ascribable
Whatever
to
proves
of of
obviating
itself
handle a
serum
a
can
serum
of
which
thousands
This
work
be
of
aided
by
antisera
pigs
guinea Sephadex
G-200
fraction. in
case
and
their
In
the
this
of
We
that the
level
15
were
No. CRN
antibody
connection,
guinea
do
thought
above
11
in anti-
24).
we
antibody No.
pig was
that
21,
first,
be
guinea This
(13,
At IgM
IgG
late
indicating
sera
of
that
the
could contributing
antibody.
type-specific
the
phenomenon pig
may
antisera
practical
may be
pigs
would
late
a grant
from
the
the
we
are
IgG
re-
with antibody
enabled
of
Ministry
ease. the
as
present
and
in
pigs
are
Secondly, in
1 :160
the
of Health
guinea
animals
used, a
present
high
system,
Furthermore,
of by
high neutrali-
because
procedures. dilution
averagely
microplate
number
type-specific
a
be
large
accomplished
at
valid,
present First,
a
absorption
used
be the
importance.
as
or
in
when
be
used
be
strains
that
anti-HSV-1
especially
can
of
was
handicaped
guinea
fractionation
available
fact
phenomenon.
(23)
the
of
rabbits
standard
such laborious
are
to
than
method
Another
properties.
for
ability
to
selection
in
explanation
rabbit
through
report
present
was
with
sera
physicochemical
type-differentiating test
much
filtration
al's
types cross-reacting
this
late
the
of
than
predominantly et
HSV
level
have
by antisera.
workers
the
gel
typing
of low
for
when
Shinkai
IgM
dilution
to
but
of
easier
for unabsorbed
other
happened
resided
sembles
on
the virus
of
explanation
by
reminded
typing
per
present
inhibition
cross-reactive
plausible
antibody,
activity
sera
with
utilizable
a
of
chromatographed
are
in
neutralization
upon
differentiation showed
were
any
guinea
zation
105
microplate
nonspecific
dilution
experiences
sera have
IgG
mere
HSV-2
contrast
of
the
done for
the
reaction
sera
by
against
the
than
continues
increased
type-specific easily
the
not
in
after
but
was antiserum
underlying
425
HERPESVIRUS
reason.
provided
sera
end
isolates
higher
idea that
OF
rabbit
titering
systems, cycles.
the
to
not
of
diluted
The
consider
does
neutralization growth
1 :20.
series.
antibody
titration
a
strains
antiserum
observation. of
NEUTRALIZATION
if
the
anti-
present
data,
method.
Welfare
of Japan
for
research
children.
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Requests for reprints should be addressed to Dr. Aiko Tada, Department of Maternal and child Health, School of Health Science, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
