Kinetics of inactivation of African swine fever antigen with binary ethylenimine


Schloer, G.M.

Proceedings of the United States Animal Health Association 86: 253-260

1982


African swine fever (ASF) soluble antigen for routine diagnostic tests is produced from infected Vero cells. The residual infectivity of antigen after solubilization and ultracentrifugation ranges from 106.5 to 108.0ID50/ml. Antigen shipped from Plum Island Animal Disease Center has to be completely inactivated, while still retaining antigenicity. Since binary ethylenimine (BEI) inactivates nucleic acid with minimal effect on protein, it was used to inactivate the residual virus present in soluble ASF antigen. The effect of time, temperature and concentration of BEI was studied. Inactivation with 10 mM BEI at 37 degrees C for 3 hours completely inactivated the virus, but some loss of antigenic activity occurred. Inactivation with 10mM BEI for 20 hours at 17 degrees C assured complete loss of infectivity while still retaining most of the antigenic activity.

KINETICS
OF
INACTIVATION
OF
AFRICAN
SWINE
FEVER
ANTIGEN
WITH
BINARY
ETHYLENIMINE
G.
M.
Schloer,
Microbiologist
United
Stated
Department
of
Agriculture
Agricultural
Research
Service
The
Plum
Island
Animal
Disease
Center
P.O.
Box
848
Greenport,
New
York
11944
ABSTRACT
African
swine
fever
(ASF)
soluble
antigen,
used
for
routine
diagnostic
tests,
is
produced
from
infected
Vero
cells.
The
residual
infectivity
of
antigen
after
solubilization
and
ultracentrifugation
ranges
from
10
6
.
5
to
10
6
11)
0
/ml.
Antigen
shipped
from
Plum
Island
Animal
Disease
Center
high
containment
laboratory
has
to
be
completely
inactivated
while
still
retaining
antigenicity.
Since
binary
ethylenimine
(BEI)
inactivates
nucleic
acid
with
minimal
effect
on
protein,
it
was
used
to
inactivate
the
residual
virus
present
in
soluble
ASF
antigen.
The
effect
of
time,
tem-
perature
and
concentration
of
BEI
was
studied.
Inactivation
with
10
mM
BEI
at
37C
for
3
hours
completely
inactivates
virus,
but
some
loss
of
antigenic
activity
occurred.
Treatment
with
10mM
BEI
at
17C
inac-
tivated
of
5
logs
of
virus
after
5
hours
of
treatment,
while
5.0
mM
BEI
inactivated
5
logs
of
virus
in
7
hours.
Inactivation
with
10mM
BEI
for
20
hours
at
17C
was
used
to
assure
complete
loss
of
infectivity
while
still
retaining
most
of
the
antigenic
activity.
Inactivated
antigen
was
found
to
contain
no
residual
infections
ASF
virus
as
demonstrated
by
inoculation
of
swine
and
tissue
cultures.
INTRODUCTION
Soluble
African
swine
fever
(ASF)
antigen
is
currently
used
for
both
the
immunoosmoelectrophoresis
(IEOP)
(4),
and
enzyme-linked
immuno-
assay
(2).
Large
scale
production
of
antigen
was
initiated
at
the
Plum
Is-
land
Animal
Disease
Center
(PIADC)
as
a
result
of
the
simultaneous
re-
port
of
outbreaks
of
ASF
in
Brazil
and
the
Dominican
Republic
in
1978
(3).
Over
10
liters
of
antigen
have
been
prepared,
inactivated,
safety
tested
and
stored
at
-70C
and
are
available
for
shipment.
Antigen
has
been
ship-
ped
both
to
the
Dominican
Republic
and
Haiti
for
laboratory
use
in
order
to
monitor
swine
sera
during
the
ASF
erradication
programs
in
those
countries.
All
biologicals
shipped
from
PIADC
must
be
free
of
infectious
agents,
thus,
complete
inactivation
of
antigen
is
mandatory
before
shipment.
Acetylethylenemine
(AEI),
B-propiolactone,
and
glycidaldehyde
have
been
shown
to
effectively
inactivate
ASFV
(7),
but
the
compounds
are
considered
potentially
carcinogenic
for
humans.
Binary
ethylenimine
(BEI)
may
also
be
potentially
carcinogenic,
but
is
is
generated
from
bromethylamine
(BEA),
a
less
toxic
compound
(1).
Previous
work
had
tied
253
254
SCHLOER
shown
that
ASF
antigen
treated
with
10
mM
BEI
for
three
hours
at
37C
completely
inactivates
the
residual
virus
in
the
antigen,
but
does
result
in
some
loss
of
antigenicity
(6).
Moreover,
storage
of
antigen
at
tein-
peratures
above
70C
also
reduces
antigen
titer.
The
kinetics
of
inac-
tivation
of
ASF
antigen
was
studied
in
order
to
establish
optimal
con-
ditions
of
antigen
inactivation
and
improve
the
quality
of
the
antigen.
MATERIALS
AND
METHODS
Virus
and
cell
culture.
The
twelfth
passage
of
ASFV/Brazi1/78
virus
adapted
to
Vero
cell
cul-
tures
was
received
from
I.
C.
Pan
of
the
PIADC.
Serial
passages
of
virus
were
made
as
described
previously
(6).
A
microtiter
assay
in
Vero
cells
was
used
to
titrate
the
virus
infectivity.
Serial
ten-fold
dilutions
of
virus
were
employed
using
four
replicates
per
dilution.
Titers
were
expressed
in
ID
50
.
Conditions
of
the
assay
will
be
described
(manuscript
in
prepara-
tion).
Immunoosmoelectrophoresis
and
immunodiffusion
assays.
Details
of
the
assay
were
described
previously
(4,
6).
All
samples
were
tested
with
a
hyperimmune
serum
obtained
from
a
pig
inoculated
with
ASFV/Dominican
Republic/78
and
ASFV/Lisbon
60.
Serum
was
obtained
from
A.
H.
Dardiri
of
the
PIADC.
Samples
for
assay
included
the
un-
treated
virus,
zero
time
and
the
final
product
after
each
treatment.
Block
titrations
of
antigen
and
antisera
were
done
for
the
IEOP
assay,
while
for
the
immunodiffusion
(ID)
assay
serial
two-fold
dilutions
of
antigen
were
tested
against
a
single
concentration
of
antiserum.
Soluble
ASF
antigen.
The
production
of
ASF
antigen
was
described
previously
(4,
6).
In
brief,
Vero
cells
grown
in
110
x
285
mm
glass
roller
bottles
were
inoculated
with
virus
at
a
multiplicity
of
infection
ranging
from
0.5
to
1.0.
After
a
4
hour
adsorption
period
at
37C,
infected
cells
were
incubated
at
33C
for
40
to
44
hours.
ASF
antigen
was
obtained
from
sonicated
infected
cells.
Af-
ter
sonication,
the
supernatant
fluid
was
clarified
by
low
and
high
speed
centrifugation.
The
final
supernatant
fluid
was
diluted
with
phosphate
buffered
saline,
(PBS)
aspH
7.4,
and
the
antigen
was
stored
at
-70C
until
it
was
activated.
The
antigen
before
inactivation
has
a
residual
virus
titer
ranging
from
10
65
to
10
80
ID-,
The
protein
concentration
was
5
mg/ml.
Determination
of
the
optimal
temperature
of
virus
inactivation
The
rate
of
inactivation
of
residual
ASF
virus
present
in
antigen
in
the
presence
of
10
mM
BEI
was
determined
at
37C,
27C,
17C
and
7C.
The
pro-
duction
of
BEI
from
BEA
was
described
previously
(1).
In
brief,
a
solution
of
100
mM
BEA
in
200
mM
NaOH
was
heated
at
37C
for
1
hour
to
produce
100
mM
BEI.
Two
control
tubes
containing
200
mM
NaOH
and
respec-
tively,
were
also
made.
Three
flasks
of
antigen
and
three
tubes
con-
taining
100
mM
BEI,
200
mM
NaOH
or
PBS,
respectivelyn
l
wriv
i
Nteri
equilibrated
for
30
minutes
at
the
above
temperatures.
At
zero
Cgree
h
b.y
4L
m
Subject
US]
AFRICAN
SWINE
FEVER
ANTIGEN
255
ml
of
BEI
was
added
to
9
ml
of
antigen
in
a
flask,
followed
by
the
addition
of
1.0
ml
of
200
mM
NaoH
or
1.0
ml
of
PBS
to
each
of
the
respective
flasks.
Flasks
were
agitated
by
stirring.
Inactivation
of
virus
by
BEI
was
stopped
by
the
addition
of
an
excess
of
Na2S
2
0,
Accordingly,
0.9
ml
samples
from
each
flask
were
rapidly
pipetted
into
0.1
ml
of
200
mM
Na2S
2
0
3
.
Samples
were
taken
at
2
minutes
after
initial
inoculation,
considered
as
zero
time,
and
at
appropriate
intervals
thereafter.
De
termination
of
the
optimal
concentration
of
BEI.
The
antigen
was
produced
from
the
23rd
passage
of
ASFV/Brazi1/78
in
Vero
cells.
Four
flasks
containing
9
ml
of
antigen
and
4
vials
of
BEI
con-
taining
100
mM,
50
mM,
25
mM
and
10
mM
BEI
were
equilibrated
at
17C.
At
0
time,
1.0
ml
of
each
concentration
of
BEI
was
added
to
a
flask
of
anti-
gen,
which
resulted
in
four
flasks
of
antigen
containing
10.0,
5.0,
2.5,
and
1.0
mM
BEI.
Flasks
were
agitated
by
stirring.
Samples
of
0.9
ml
were
taken
from
each
flask
at
0,
2,
4,
5,
and
24
hours
post
inoculation
(p.i.)
and
diluted
into
0.1
ml
of
200
mM
Na
2
S
2
03.
Samples
were
stored
at
5C
before
assay.
RESULTS
Optimal
temperature
of
inactivation.
The
rate
of
inactivation
of
virus
by
10
mM
BEI
is
linear
and
decreases
with
the
decrease
in
temperature
at
37C,
27C
and
17C,
with
no
viable
virus
detected
at
1,
3
and
approximately
10
hours,
respectively.
Reaction
at
7C
was
slower
with
some
residual
infectivity
seen
at
24
hours
post
in-
oculation.
Antigen
diluted
in
PBS
showed
no
drop
in
titer
when
treated
at
37C
for
2
hours,
27C
for
4
hours,
17C
and
7C
for
24
hours.
In
all
cases,
anti-
gen
treated
with
200
mM
NaOH
at
the
above
time
and
temperature
was
non-infectious.
IEOP
assays
on
the
original,
zero
time
and
final
samples
after
treat-
ment
at
different
temperatures
are
shown
in
Figure
2.
There
was
only
a
minimal
effect
on
antigen
treated
with
10
mM
BEI
at
37C
and
27C
for
1.5
and
2
hours,
respectively.
Little
or
no
reduction
in
antigenicity
was
seen
in
virus
treated
with
10
mM
BEI
at
17C
and
7C.
Figure
3
shows
the
re-
sults
of
titrations
of
antigen
with
10
mM
BEI
at
different
temperatures.
There
appear
to
be
at
least
4
major
precipitin
lines
detected
in
both
un-
stained
preparations
of
the
IEOP
and
ID
assays.
The
number
of
lines
di-
minishes
with
antigen
dilution.
A
principal
antigen
had
a
titer
of
1/32
in
the
ID
assay
when
virus
was
treated
with
BEI
at
17C
and
7C.
However,
the
antigen
had
a
titer
of
1/8
when
treated
at
37C
for
1.5
hours
with
10
mM
BEI.
The
identity
of
these
principal
antigens
is
unknown
at
present.
Inactivation
with
10
mM
BEI
at
37C
and
27C
is
extremely
rapid,
whereas
inactivation
at
17C
was
effective
and
readily
controlled.
This
temperature
was
chosen
as
optimal
for
inactivation.
Optimal
concentration
of
BEL
s
copied
lay
be
he
rate
of
inactivation
at
17C
with
varying
concentrations
of
BEI
is
ight
Laws
256
SCHLOER
seen
in
Figure
4.
Inactivation
with
1.0
mM
BEI
was
slow
with
no
inactiva-
tion
occurring
at
5
hours
p.i.
Some
inactivation
not
indicated
first
detected
was
was
seen
at
24
hours
p.i.
Inactivation
of
virus
with
2.5
mM
detected
at
4
hours
p.i.
Treatment
with
10
mM
BEI
showed
inactivation
in
the
figur
e
t
Art
RE!
inactivated
of
5
logs
of
virus
after
5
hours
of
treatment,
while
5.0
Tri
,
5
logs
of
virus
in
7
hours.
a
n
cti
can
va
.
The
resid
ua
l
v
i
rus
titer
in
different
lots
of
soluble
ASF
aritige
range
from
10
6
.3
to
106.0
ID
50/ml.
Most
lots
of
antigen
prepared
fort%
ora-
tion
represent
a
pool
of
approximately
360
ml.
In
order
to
as
sai
.,
mM
in
ac
ti
vation
of
residual
virus,
antigen
was
inactivated
with
lu
BEI
for
20
hours
at
17C.
DISCUSSION
Complete
i
nact
i
vation
of
residual
virus
present
in
soluble
ASF
antigen
is
necessary
b
ecause
the
antigen
is
shipped
both
to
Latin
Arnerica
tries
and
to
th
e
N
ational
Veterinary
Services
Laboratory
at
Ar
ne
ve
been
Over
15
lot
s
o
f
A
SF/Brazil/78
antigen,
400
ml
of
antigen
per
lot,
119
_
te
lass
treated
by
th
e
a
b
ove
method.
All
of
the
lots
have
shown
a
complet
toad
e
of
infectivity
as
indicated
by
inoculation
of
both
animals
and
Aso
American
,
cultures
(unp
u
bli
s
h
ed
data).
Although
5
mM
BEI
treatment
may
c
u
in
effective,
tre
a
t
ment
with
10
mM
BEI
for
20
hours
at
17C
results
wtore-
minimal
effect
on
antigenicity
obvbetr,
repeated
tests
on
antigen
stored
at
-
70C
for
as
long
as
2
,
(an-
and
assures
inactivation
of
Years
virus.°'
a
small
decrease
in
antigenicity
as
measured
by
the
IEOP
a
ss
'
published
data).
lira
can
+
African
swine
fever
virus
is
a
extremely
stable
virus,
which
,„
w
thau
stand
A
s
r
v
wide
extremes
of
temperature
and
pH
(5,
8).
Our
results
sn
t
"
ion
of
is
rapidly
inactivated
by
20
mM
NaOH
at
a
final
pH
in
antigen
era
ains
9.0.
bfet.
This
differs
from
previous
reports
which
indicate
that
ASF
v
r
of
e
mus
after
treatment
for
6
hours
at
pH
13.0.
Both
the
total
amoun
t
virus
protein
present
in
the
preparation
and,
perhaps,
differences
among'
strains
may
account
for
the
discrepancy
in
results.
is
aYs
The
number
of
precipitin
lines
seen
both
by
the
IEOP
andID
assnage°
an
indication
of
the
multiplicity
of
antigens
present
in
the
ASF
ears
e
paration.
It
is
interesting
to
note
that
one
of
the
antigens
aPP
o
f
the
sensitive
to
treatment
at
37C.
The
identification
and
isolation
a
n
jar
principle
viral
proteins
that
react
in
the
IEOP
assay
will
result
in
proved
and
more
specific
test.
Inac
tivation
of
r
es
id
ual
ASF
virus
in
antigen
with
10
mM
BEI
a
n
i
mal
for
24
hours
effectively
inactivates
infectivity
of
ASF
virus
with
In
effect
on
viral
antigenicity.
ACKNOWLEDGEMENTS
The
exc
e
ll
ent
technical
assistance
of
David
Perkins
and
Elizabeth
King
is
gratefully
acknowledged.
This
m
ateri
a
at
the
NLM
a
Subject
UL.Sidi
AFRICAN
SWINE
FEVER
ANTIGEN
257
100
.
0
10.0
0
0
1.0
cc
co
LL
tc
0
.
1
0.01
37
°
c
17
0
c
A
27
°
c
1
2
3
4
5
HRS.
in
10
mM
BEI
r
igu
0
-•
—omparison
of
the
effect
of
different
temperatures
on
the
inac-
""
of
ASP/Brazil/78
antigen
treated
with
10
mM
BEI.
AFRICAN
SWINE
FEVER
ANTIGEN
AG
'AG
1/1
259
1/32
1/32
ii
/
11
1
0
/k
1/2
27°C
AB
1/1
AB
1/1
37°C
i/16
1
/
1
6
1/4
1/8
1/8
AG
1/1
AG
1/1
/C;2
1/2)
1/32
1/2
7°C
AB
1/1
AB
1/1
17°C
1/16
1/4
1/16
1/4
1/8
1/8
Figure
3.
Immunodiffusion
titration
of
ASF
antigen
with
hyperimmune
anti-ASFV
serum
as
described
in
Figure
2.
100.0
1.0
mM
BEI
10.0
2.5
mM
BEI
5.0
mM
BEI
10.0
mM
BEI
2
4
<.01
6
HR
at
17
°
c
ed
Figure
4.
Comparison
of
the
effect
of
different
concentrations
of
BEI
on
the
inactivation
of
ASFV/Brazil/78
at
17°C.
3WS
ASFV
SU
RVIV
AL
L
OG
1
o
1.0
0.1
0.01
260
SCHLOER
REFERENCES
1.
Bahnemann,
H.:
Binary
ethylenimine
as
an
inactivant
for
foot-and-mouth
disease
virus
and
its
application
for
vaccine
production.
Archly.
Virol.
47,
(1975):
47-56.
2.
Hamdy,
F.M.
and
Dardiri,
A.H.:
Enzyme-linked
immunosorhent
assay
for
the
diagnosis
of
African
swine
fever.
Vet.
Rec.
/05,
(1979):
445-446.
3.
Mebus,
C.A.,
Dardiri,
A.H.,
Hamdy,
Ferris,
D.H.,
Hess,
W.R.
and
Callis,
J.J.:
Some
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