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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Enzyme-linked immunosorbent assay for efficient detection of antibody to bluetongue virus in pronghorn (Antilocapra americana).

An indirect enzyme-linked immunosorbent assay (ELISA), using cell-associated viral antigen, was developed for detection of antibody to bluetongue viru...
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