ENZYME-LINKED IMMUNOSORBENT ASSAY FOR EFFICIENT DETECTION OF ANTIBODY TO BLUETONGUE VIRUS IN PRONGHORN (ANTILOCAPRA AMERICANA) Author(s): B. S. Drolet, K. W. Mills, E. L. Belden, and J. O. Mecham Source: Journal of Wildlife Diseases, 26(1):34-40. Published By: Wildlife Disease Association DOI: http://dx.doi.org/10.7589/0090-3558-26.1.34 URL: http://www.bioone.org/doi/full/10.7589/0090-3558-26.1.34
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Journal
ENZYME-LINKED DETECTION
IMMUNOSORBENT OF ANTIBODY
PRONGHORN B. S. Drolet, 2
of Veterinary
Department
E. L. Belden,2
An
ABSTRACT:
ic studies,
The
serum
results
samples numbers
Key
BTV
ELISA
of
sera
from reaction
gave
for
BT
rapid,
Bluetongue
(BT) disease Pronghorn are
is an
(ELISA),
and wild (Antilocapra affected
bluetongue virus (BTV) (Thorne 1988) and are an important source formation in studying BT antibody alence where pronghorn grazing overlap stock.
livestock (Trainer Diagnosis of marily
on
anti-BTV Bluetongue (Pearson
studies. assay’s 1987; results, ity of
in wild of the
and Jochim, BTV infection
detection
of
antibodies
Della-Porta
USDA export
BT that
ru-
et
by
a!.,
ruminants disease
and
Jochim,
False negative limited sensitivity Poli et a!., 1982), thought to be the antigenically
hemorrhagic shar et a!.,
should
virus.
facilitate
testing
indirect
en-
study.
viral
antigen for the detecto BTV in field-collected The
samples
also
AND MATERIALS
viral antigen
hamster in
were
CA antigen to reactions caused
850
kidney cm2
roller
cells bottles
(BHK-21/69) (Corning,
El-
mira, New York 14902, USA) were infected with either cell culture-adapted BTV serotype 11 (BTV-11) or EHDV serotype 2 (EHDV-2) (USDA-ARS, Laramie, Wyoming 82071, USA) at 0.024 plaque forming units per cell. Cells were harvested after 3 days (near maximum cytopathic effect) by centrifugation at 1,000 g for 15 mm. Cell-associated antigens were pre-
pri-
official
pared
Briefly, pended
results, due to the (Afshar et al., and false positive
cated
essentially
as described
by Jochim
cells were washed, harvested, in tris buffer (0.002 M, pH in an
ice
water
bath
and
(1976).
twice 8.8),
centrifuged
sussoniat
g. The antigen protocol was modified at this point in that the resulting supernatants were pooled, twice vortexed with Fluorocarbon 113 (‘/3 volume), and centrifuged at 1,000 g to eliminate cellular lipids. Aqueous phases were pooled and assayed for protein concentration (Bio-Rad, Richmond, California 94804, USA). For the BHK 2,300
to cross-reactivsimilar epizootic
disease virus (EHDV) 1987; White et a!., 1985),
field
sera.
Baby
sera for import! in epizootiologic
due
Discrepant disease
seroepizootiology,
test,
grown
1985).
is the
and
MATERIALS
(BTID)
1979),
assay for testing and is often used
Bluetongue
assay.
studies.
Cell-associated
1985;
Jochim,
tested.
hemorrhagic
results
pronghorn
group-specific
1985;
were
tested against uninfected determine if background false positive results.
in
1969). relies
immunodiffusion
epizootic
sociated (CA) tion of antibodies
et a!., of inprevareas
(Anderson,
or
viral
make the BTID unreliable. Described herein is an indirect enzyme-linked immunosorbent assay (ELISA), using cell-as-
those of domestic ruminant liveIt has been shown that the geograph-
ical distribution of generally parallels
cell-associated
(BTV) in field-collected ELISA to seroepizootiolog-
(USA)
bluetongue,
diagnostic
P.O. Box 3965, Uni-
immunodiffusion
seroepizootiologic
americana,
using
of the
counties
BTV
Laboratory,
virus
objective
arthropod-borne
of domestic antelope commonly
IN
(ELISA),
bluetongue
either
and
Research
applicability
Wyoming
INTRODUCTION
orbiviral minants. americana)
j4-4U
82071, USA 82071, USA
Wyoming
Wyoming
Diseases
quantitative,
diagnostic assay
VIRUS
assay
of the to
Antilocapra
immunosorbent
pp.
1990,
EFFICIENT
to bluetongue
test the
three
to those
for
Pronghorn,
Animal
of antibody sera. To
compared
retested
words:
zymne-linked
were
Laramie,
Laramie,
immunosorbent
detection samples
were
pronghorn
large
for
americana)
pronghorn
ELISA
serum
enzyme-linked
(Antilocapra
pronghorn
virus
indirect
developed
was
26(1),
and J. 0. Mecham34
3USDA Agricultural Research Service, Arthropod-Borne versity Station, Laramie, Wyoming 82071, USA Author to whom correspondence should be addressed
antigen,
Diseases,
AMERICANA)
Sciences, University of Wyoming, Biology, University of Wyoming,
of Molecular
FOR
TO BLUETONGUE
(ANTILOCAPRA K. W. Mills,’
Department
ASSAY
of Wildlife
(Afcan 34
DROLET
control
antigen,
scraped
uninfected
cell
monolayers
were
treated cells.
the same
from roller bottles, then BTV and EHDV infected
as the
ET AL.-BLUETONGUE
FOR PRONGHORN
ELISA
35
1600
1.400
1.200
Pronghom horseradish peroxidase (HRP)-conjugated antibody
Cl)
1000
z
Pronghorn serum was precipitated with saturated (NH4)2S04 and IgG purified on a Sephacel anion exchange column (Pharmacia, Piscataway, New Jersey 08855, USA). Rabbits were inoculated with 200 g pronghorn IgG in complete Freund’s adjuvant (50 sg subcutaneously, 50 tg intravenously, and 100 tg intramuscularly). Twenty-one and 28 days later, 200 sg IgG boosters, with adjuvant (Tetronic 1501 Polyol, BASF Wyandotte Corporation Industrial Chemical Group, Wyandotte, Michigan 49192, USA) (Hunter and Bennett, 1984; Woodard, 1989), were injected subcutaneously. After 7 days rabbits
were
bled
and
rabbit
anti-pronghorn
IgG
was
precipitated with saturated (NH4)2S04 bert et al., 1973). Antibody was conjugated horseradish
peroxidase
using
(Nakane
and
method
iodate
Pronghom
One
serologic
hundred
killed
in
verse,
and
Game ming
and 82070, BTV,
using tongue
sixty-one
or hunter-
samples
Wyoming
from
(USA)
Carbon),
provided
APHIS
at the
Wyoming
oratory
(Laramie,
(Johnson, by
were
BTV,
EHDV
were
assays performed
State
Wyoming BTID tested
and BHK
0800
0 I0.
(5
0
Wyoming
positive,
WyoELISA Blue-
(Pearson
by the
and
USDA-
Veterinary 82070, but
..J’....
#{149} : 0200
. #{149}
S
#{149} #{149}#{149}#{149}.#{149} #{149}“:
#{149}#{149}
0
170
SERUM
1.
FIGURE
samples
tested
with
the
for
BTV
of
BTID+/ELISA+
tested
positive
for
and
the ELISA.
ples
that
the
ELISA.
were
positive
in
BTID-/ELISA-
in
(+) the
in both
BTID,
BTID,
the
but
and
that
BTID
indicate but
(#{149}) indicate the BTID
assay samples
sam-
positive
indicate
(0)
the
assay
in both
BTID-/ELISA+
BTID+/ELISA-
negative
indicate
antibody
negative
(BTV)
immunodiffusion
() BTV
serum
virus
immunosorbent
bluetongue
(BTID).
pronghorn
to bluetongue
enzyme-linked
the
and
were
graph
antibody
(ELISA)
ELISA.
SAMPLES
Scatter
negative
in
samples in
samples
the
that
the ELISA.
Con-
the
Lab-
USA). BTV
ELISA
presence of EHDV antibody with the ELISA using EHDV CA antigen. Sera that were BTID negative and BTV ELISA positive were radioimmune precipitated to demonstrate serum antibodies to BT structural and non-structural viral proteins. negative
-J
three
Fish Department (Laramie, USA), were tested with the EHDV and BHK antigens. were
that
1979).
trapped
serum
1979)
Sera
Kawaoi,
0.800
that were
immunodiffusion
Jochim,
to m-per-
survey
pronghorn
counties
the sodium
(He-
a
for the
ELISA
Ninety-six well microtiter plates (Dynatech Immulon II, Chantilly, Virginia 22021, USA) were coated with the appropriate CA antigen (BTV-11 at 4 tg, EHDV-2 at 8 og or BHK at 4 g) in 100 l coating buffer (0.07 M NaHCO3, 0.03 M Na2CO3, pH 9.6) (Roehrig et al., 1980). Plates were allowed to stand overnight at 4 C in a humid chamber. Plates were washed three times with washing solution [phosphate buffered saline (PBS), 0.5% non-fat powdered milk (NPM), 0.05% Tween 20, 0.005% Thimerosal, pH 7.2] and rinsed once with distilled water between each of the steps. Incubation parameters were 100 sl/well, 1 hr at 37 C, unless
indicated otherwise. Non-specific binding sites in the plate wells were blocked with 5% milk block solution (Swack et al., 1987) (in coating buffer, 0.005% Thimerosal), 200 ol/well, 2 hr at 37 C. Pronghorn serum samples were diluted 1:100 in dilution buffer (PBS, 0.5% NPM, 0.005% Thimerosal, pH 7.2). Twenty-two samples were tested per plate, three replicate wells/sample. Rabbit anti-pronghorn IgG-HRP conjugate (1: 1,000 dilution) was then added and plates were incubated. Ortho-phenylene diamine (OPD, Hyclone Laboratories, Logan, Utah 84321, USA) (Shaw et al., 1986) was added and incubated in the dark, at room temperature for 20 mm. The enzymatic reaction was stopped with 3 N sulfuric acid (50 tl/well) and the optical density (OD) was determined with a Titertek Multiskan Plus MK II (Flow Laboratories, McLean, Virginia 22102, USA) plate reader at A492nm. Pronghorn sera were tested with a BHK control CA antigen to determine the background reactivity of the BTV ELISA to non-viral antigen. BHK control ELISA results were subtracted from the BTV ELISA results. One hundred thirty-two BTID negative sera were used to calculate the positive/negative cutoff. Antelope sera that were BTID positive/BTV ELISA negative and two serum samples that were BTID strong positive/BTV ELISA weak positive were tested for EHDV antibody with the EHDV ELISA.
36
JOURNAL
OF WILDLIFE
DISEASES,
VOL. 26, NO. 1, JANUARY
1. Results of 161 pronghorn serum as tested by the enzyme-linked immunosorbent (ELISA) and the bluetongue immunodiffusion (BTID).
1990
1600
samples assay assay
TABLE
1.400
1.200
Cl)
ELISA BTID
a
Total
(-)
(+)
1.000
z .#
-I 0
(+)
22
3
25
4
132
136
135
161
(-)
Total
26
0.600
0.
0
0.400
#{149}
#{149} #{149} L%
ELISA
170
Sensitivity
=
Specificity
=
22/25
88%
=
132/136
SERUM
Predictive
(+) value
=
22/26
Predictive
(-)
value
=
132/135
84.6%
=
=
samples
97.8%
precipitation cells were
infected with BTV-11, labelled with [S]metliionine (Du Pont, NEN, Wilmington, Delaware 19801, USA), and lysed as described by Mecham et al. (1986). Viral proteins in the cell lysates were immune precipitated using pronghorn antisera in combiIlatioll with Staphylococcus aureus protein A (Pansorbin, Calbiochem, La Jolla, California 92037, USA) as the solid phase. Immune precipitated
viral
antigens
were
(polvacrylamide autoradiograph. PAGE
gel
analyzed
by
electrophoresis)
SDSand
ELISA
Variability
in the
optical OD
density values
tive/negative dard
OD
cut
deviations
to
off:
mean
Test sera with an OD sidered negative and >0.396 were considered
+
OD
results
OD
lower a posi-
+
(3)0.067
=
3 stan0.396].
0.396 were sera with an positive.
thirty-two sera ELISA negative.
of the
BTID
The
calculate
two sera were BTID positive/BTV positive. Four sera were BTID BTV ELISA positive, and three BTID positive, but consistently ative in the BTV ELISA (Table The
of
was seen, as near 0.350 1).
(Fig.
used [0.195
One hundred negative/BTV
results
test sera the results
(OD) were
of
serum
pronghorn
to bluetongue
virus (BTV)
enzyme-linked the bluetongue minus the cellular
immunosorbent assay immunodiffusion assay (BTID), background inherent in the cell-associated antigen. BTID+/ELISA+ (4) indicate samples that tested positive for BTV antibody in both the BTID and the ELISA. BTID-/ELISA+ (+) indicate samples that were negative in the BTID, but positive in the ELISA. BTID+/ELISA(0) inthe BTV
dicate
and
samples
negative samples ELISA.
p!es
that
were
positive
in
in the ELISA. BTID-/ELISAthat were negative in both
tested
ied from
in the 0.062
BHK
to 0.381.
the
BTID,
but
(#{149}) indicate the BTID
control
and
var-
ELISA
Results
the
of the
BTV
ELISA on the 161 serum samples after subtraction of this BHK control background and calculation of the positive/negative cut off [mean OD + 3 standard devia-
RESULTS
negative pronghorn well as a break in
graph
tested for antibody
(ELISA)
BHK-2l
Scatter
2.
FIGURE
with
Radioimmune
SAMPLES
97%
=
161
conOD
were BTID TwentyELISA negative/ sera were tested neg1).
antelope
sam-
tions
(0.035
+
(3)0.049
equivalent to the results of the BHK background
=
0.182)]
before (Fig.
were
subtraction 2).
Three known BTID negative pronghorn serum samples, three OD readings/sample, were used for the calculation of a positive/negative cut off of 0.119 [0.095 + (3)0.008 = 0.119] for the EHDV ELISA. One of the three BTID positive/BTV ELISA negative samples was positive for EHDV antibody, and the BTID strong positive/BTV ELISA weak positive samples were both positive for EHDV antibodies by
the
EHDV
ELISA
(Fig.
3).
The 1988 estimated postharvest wintering pronghorn population in Wyoming was 363,487 animals (Wyoming Game and Fish,
1988).
Of
161
antelope
samples
from
DROLET
ET AL.-BLUETONGUE
1000
12
ELISA FOR PRONGHORN
34
37
56789 4
0800 >.
ICl)
z
0600
60
a 4 0 I-
VP1
0400
0.
0
A
VP 2/3
0200
A
QD#{216}
VP4
0000
0
10
SERUM
FIGURE tested
disease
(EHDV)
virus
immunosorbent but
weak
()
and
an
positives
the
VP7
BTID+/
positive
positive
the
in
ELISA.
but negative
t.
#{149}‘
VP6
that were
negative
EHDV
NS2
+/ELISA+/positives
___
NS1
enzyme-linked
strong in
serum
hemorrhagic
BTID+
samples
EHDV
known
pronghorn
EHDV
were
in the BTID,
known two
that
indicate
antibody
Three
with
samples
ELISA-
of
to epizootic
assay (ELISA).
(A) indicate BTID,
graph
antibody
for
VP5
SAMPLES
Scatter
3.
samples
BTV
-
-
OFF
4-+/-CUT
A
8050*
for
in the ELISA.
control
antisera
(0)
control
antisera
(#{149})
are also shown.
three
Wyoming
were
positive
BTV
ELISA.
counties,
for
Radioimmune
discrepant positive
demonstrated
to
immune
rum ative teins
1986) (Fig. positive/BTV
(16%)
with
the
have
(Fig.
5).
BTV
antibodies
structural of
BTV
Non-specific
and
as in a known
was
seen
negative
of
Radioimmune antigen
from
infected
se-
sera;
except
sera, at
radioimmune
control
positive
seven
dilution, serum
were samples.
proteins
[aSimethionine,
lane
1, rabbit
antiserum
were antiserum
to BTV-11;
7, pronghorn
lanes negative
positive/BTID antiserum
BTV;
to
labelled cell lysate; lane 9, normal
precipitation. sera
for
were
of
positive ELISA,
the
three
indicate
the
BTID,
or
The
was
BTID.
may
viruses
sera
result
positive
other two
in the
non-specific cross-reactive
than sera
neg-
proteins
by
precipitation.
antibody account
sera
BTID
to be
BTV
for EHDV which would
equivalent
negative sera were demonstrated to be positive for antibodies to BTV structural and non-structural proteins by BTV ELISA and
to
radioimmune
Four
BTID
Three
demonstrated
antibodies and
One
of the BTID, which requires and ELISA, which tests sam-
a 1:100 for
of cell-asViral
the
in these
DISCUSSION
ples
sera.
serum.
ELISA
whole
with: weak
lane
lane 8, uninfected
ative
results
ELISA
test
with
lane 2, bovine
to BTV-11;
pronghorn
precipitation with
labelled
precipitated
pronghorn
sera.
The
cells,
3 to 6, BTV
to be negviral pro-
sticking NS1
4. BTV-11
FIGURE
sociated immune
(Mecham
demonstrated to structural
protein
we!!
negative/ samples were
4). The three discrepant ELISA negative
samples were for antibodies
as
serum
proteins
nonstructural sera
BTID
precipitated
nonstructura!
et a!., BTID
samples
antibody
precipitation
The four BTV ELISA which
26
BTV
shown
to
by for The
be
EHDV a false
other
two
reactions
in
antibodies
to
EHDV. that
were
strong
BTID
38
JOURNAL
OF WILDLIFE
DISEASES,
1234
VOL
26, NO
56
“
7
1, JANUARY
8
1990
fected
9
by
the
presence
of EHDV
antibod-
ies may be due to the slightly altered reactivity of cross-reactive antibodies to antigen adsorbed to polystyrene (Butler et a!., 1986; Dierks et a!., 1986). Another explanation is that the cross-reacting antibodies causing precipitation in the BTID are possible
vp1
low-affinity
antibodies
which
would
not
be
detected in the ELISA (Butler et a!., 1978). Four additional positive samples along with one false BTID positive sample
VP2’3
VP4
(EHDV
positive)
were
been
false
NSI
senting an improvement Cellular background antigen had negligible when a positive/negative
over the BTID. reaction in the CA effects on the results cut off was cal-
culated with + 3 standard
negative to give
negatives
in the
a known deviations
interval. is simple,
samples
The CA economic
may
the
ELISA,
fidence ration
two
with
BTV
N52 VP6 VP7
while
discovered
VP5
ELISA,
have repre-
OD mean a 99% con-
antigen prepaand high-yield-
ing. The ELISA
increased can test
in
number one
the BTID is substantial. ples, three replicate run on a pre-coated 5.
FIGURE
sociated
Radioimmune 1 antigen
BTV-1
from
infected
cells,
labelled
precipitated:
immune belled
cell
lysate;
lane
control
pronghorn
known
positive
negative
cell lysate;
lysate
of cell-as-
Viral
were
infected,
lysate
sera;
lane
with
sera;
positive/BTV
control
pronghorn
with
known
positive
sera;
lane
labelled
known neguninfected lysate with
lysate
control
lylane
ELISA
sera; lane 7, uninfected,
ative
laknown
3, infected
pronghorn
BTID
8, uninfected
proteins
[‘SJmethionine,
1, BTV-11
control
with
pronghorn
lane
sera.
2, infected
negative
4 to 6, infected
test
with
lane
sate
with
precipitation with
9,
pronghorn
sera.
positives, but weak BTV ELISA positives, were shown to have EHDV antibodies by EHDV ELISA, as suspected. An animal infected with both BTV and EHDV would give a stronger which is affected
positive reading by BTID by EHDV cross-reactive
antibodies, than which specifically
it
would detects
in the ELISA BTV antibodies
only. The
reason
the
BTV
ELISA
is not
af-
of day
samples compared
the to
Twenty-two samwells/sample can be plate in about 5 hr.
Pre-coated plates can be stored at 4 C in a humid chamber for up to 10 wk. Several plates can be run simultaneously and semimechanization is possible, which lends itself well to testing large numbers of samples relatively quickly. The BTID can only test three samples per assay and must be incubated 24 hr before reading. The pronghorn BTV ELISA was adapted by this laboratory from a bovine BTV ELISA by the substitution of one reagent-the anti-species IgG-HRP conjugate. This ELISA also has been adapted to deer and elk by another laboratory with the appropriate anti-species conjugate substitution. It is probable that the ELISA could easily be adapted to moose, bighorn sheep and other wildlife. The quantity and quality of serum samples obtained from wildlife are often less than optimal. The BTV ELISA requires only
3 l of serum
for
testing
as described
DROLET
herein, gardless
and of
storage
of
will prior the
give accurate improper sample.
results handling
The
BTV
ELISA
gave rapid, quantitative, objective and should prove useful in testing numbers of sera for BT diagnostic roepizootiologic
reor
results large and se-
ET AL.-BLUETONGUE
ELISA FOR PRONGHORN
39
bit, sheep, horse, and goat antisera. Applied Microbiology 25: 26-36. HUNTER, R. L., AND B. BENNETT. 1984. The adjuvant activity of nonionic block polymer surfactants. II. Antibody formation and inflammation related to the structure of triblock and octablock copolymers. Journal of Immunology 133: 3167-3175.
studies.
M. M. 1976. Improvement of the AGP test for bluetongue. Proceedings of the American Association of Veterinary Laboratory Diagnosticians 19: 361-376. 1985. An overview of diagnostics for bluetongue. In Bluetongue and related orbiviruses, T. L. Barber and M. M. Jochim (eds.). Alan R. Liss, Inc., New York, New York, pp. 423-433. MECHAM, J. 0., V. C. DEAN, AND M. M. JOCHIM. 1986. Correlation of serotype specificity and protein structure of the five serotypes of bluetongue virus. Journal of General Virology 67:
JOCHIM,
ACKNOWLEDGMENTS We
acknowledge
Thorne
and
the
the
assistance
Wyoming
of
Game
and
E.
T.
Fish
De-
partment for providing the pronghorn sera necessary for the development of this assay. We also acknowledge the United States Department of Agriculture-Animal and Plant Health Inspection Service for performing the bluetongue immunodiffusion assays. LITERATURE A.
F. C., P. F.
SHAPIRO,
P. T.
AFSHAR,
L.
1987.
2617-2624.
P. F.
SHETTIGARA,
Comparison
tion of bluetongue whole blood. Journal
ANDERSON.
indirect
and
immunosorbent
J.
WRIGHT,
J.
AND
of competitive
enzyme-linked
1705-
CITED
THOMAS,
assay
for detec-
virus antibodies in serum of Clinical Microbiology
and 25:
1710.
J.
ANDERSON,
1985.
bluetongue
Monoclonal
virus
related
orbiviruses,
chim
(eds.). Alan
R. Liss,
York,
pp. 497-504. J. E., T. L.
FELDBUSH,
BUTLER,
N.
AND
T.
L.
and
Inc.,
P. L. The
M.
and Jo-
M.
York,
New
assay (ELISA):
concentration
and
Bluetongue
Barber
1978
STEWART.
immunosorbent tibody
antibodies
In
diagnosis.
New
tection
MCGIVERN,
enzyme-linked A measure
of an-
or affinity? Immunochem-
istry 15: 131-136.
J. E.
M.
SPRADLING,
HEYERMANN,
AND
immunochemistry binding
J.
S. E.
of sandwich
characteristics
monoclonal adsorbed metrical conjugates.
SUTER,
H. PETERMAN.
H.
DIERK5,
1986.
The
ELISAs-I.
The
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