Antigen capture elisa for the identification of dengue viruses

Journal of Virological Methods, 12 (1985) 93-103

93

Elsevier JVM 00438

ANTIGEN

CAPTURE

ELISA

FOR THE IDENTIFICATION

OF DENGUE

VIRUSES

G. KUNOI,

D.J. GUBLER’

and N. SANTIAGO

DE WEIL2

‘Dengue Branch, Division of Vector-Borne Viral Diseases,

Centerfor Infectious Diseases, Centersfor Disease

Control, San Juan, and =Department of Biology, University of Puerto Rico, Rio Piedras, Puerto Rico (Accepted

20 June

We developed

1985)

a simple

dengue (DEN) viruses

antigen

The method

ies and an enzyme conjugate DEN virus strains,

capture

enzyme

employed

of a tlavivirus-reactive

representing

all 4 serotypes

either visually or spectrophotometrically

dengue

virus

antibodies

antibody

from various

with monoclonal

after storage

virus identification

monoclonal

isolated

assay (A&-ELISA]

monoclonal

when infected cell culture

The shelf life of the solid phase presensitized type viruses were still identified

immunosorbent

serotype-specific

for identifying

as capture

as a detecttng

parts of the tropics,

antibod-

antibody.

Most

were identified

fluids were used as sources ofantigen.

antibodies

was 4 mth at - 15°C. DEN proto-

at -15OC for 1 yr or at room

temperature

for 1 mth.

ELISA

INTRODUCTION

Dengue (DEN) viruses have been among the most difficult of the arboviruses to identify because they do not replicate to high titer in most mammalian cell cultures and they belong to a group of serologically related viruses which cross react in most tests. The most reliable

identification

tion test (PRNT)

using LLC-MK2

ment fixation

techniques

(CF) test using antigen

1977). Both tests use polyclonal

have been the plaque reduction

cells (Russell and Nisalak, prepared

antibodies,

in mosquitoes

usually

produced

neutraliza-

1967) and the comple(Kuberski

and Rosen,

as ascitic fluids in mice.

More recently, serotype-specific monoclonal antibodies became available for dengue viruses (Gentry et al., 1982) and identification was simplified by the use of a monoclonal antibody indirect fluorescent antibody (IFA) test (Henchal et al., 1982; Gubler et al., 1984). Enzyme-linked immunosorbent assay (ELISA) has gained popularity for use in diagnosing viral diseases because of its simplicity, rapidity, and economy (Yolken, 1982), as well as its high sensitivity and specificity. Antigen capture ELISA (AgC-ELISA) has been successfully used to identify many viruses. Here we report the use of AgC-ELISA to identify DEN viruses.

94

MATERIALS

ELISA

AND METHODS

procedure for virus identification

The procedure Monath

was a modification

and Nystrom

of one developed

(1984). Immulon

for yellow fever (YF) viruses by

II plates (Dynatech

VA)* were rinsed once in hot water and air-dried

Laboratories,

before sensitization.

Alexandria, The central

60

wells were demarcated vertically into 2 equal fields (right and left),each field containing 30 wells (5 columns X 6 rows). Monoclonal antibodies for each dengue serotype were diluted in a carbonate buffer (pH 9.6), and 100 ul aliquots were dropped into each well of a column (1 column/field/monoclonal antibody). Aliquots (100 ~1) of normal mouse ascitic fluid were dropped into wells of the fifth column. A phosphate-buffered saline

(PBS) containing

0.05% Tween

20 (PBS-0.05%

T; pH 7.5) was added to the

unused marginal wells (50 ul/well). The plates were incubated in a humidified tray overnight at 4°C and washed 5 times with PBS-0.05% T. Prototype DEN suspensions prepared in suckling mouse brain (20%) and/or TRA284-SF mosquito cell cultures (Kuno, 1982) were used as antigen undiluted ordiluted in the PBS containing 0.1% Tween 20 (PBS-0.1% T; pH 7.5). All nonprototype DEN strains

tested had been previously

monoclonal antibodies (Gubler infected mosquitoes (Kuberski neutralization 1967).

test using

identified

by the IFA test using serotype-specific

et al., 1984), by CF test using antigen prepared from and Rosen, 1977), and/or by 50% plaque reduction

rhesus

monkey

kidney

cell cultures

(Russell

and Nisalak,

Only the supernatant fluids from infected mosquito cell cultures were used as antigen for the nonprototype virus AgC-ELISA tests. All viruses had been frozen at -65°C or lower with 20% heat-inactivated bovine calf serum. Each unknown virus plus the 4 prototype control viruses were dispensed (100 ul/well) into the wells of a single row (1 row/virus).

The last row of each plate was the negative

control,

the supernatant fluid of normal cell culture. The plates were incubated A maximum of 11 viruses could be tested per plate. The alkaline

phosphatase

conjugate

was prepared

clonal antibody (6B6C-1) by the Division Centers for Disease Control at Ft. Collins, The conjugate,

diluted

1 : 320 with PBS-0.1%

from a flavivirus

inoculated

-reactive

of Vector-Borne Viral Diseases Colorado (Monath and Nystrom, T, was dispensed

with

for 3 hat 36°C.

(100 ul/well)

monoof the 1984). into the

central wells after the plates had been rinsed 5 times. The plates were incubated at 36°C for 2 h. Finally, 100 ul of a Tris buffer (pH 8.0) substrate solution containing 3 mgp-nitrophenyl phosphate per ml was added to each of the central wells, and the plates were incubated at room temperature (RT: 22-23°C) for up to 6 h. The color reactions were read visually at 1,3, and 6 h, and spectrophotometrically using an ELISA Mini-Reader

*Use of trade names and commercial the Public Health

sources

is for identification

Service or the U.S. Department

of Health

only and does not imply endorsement

and Human

Services.

by

95

II (Dynatech visually

Laboratories),

identified

absorbance

at 405 nm 3 h after addition

by selecting

the wells that demonstrated

values were obtained

spectrophotometrically.

bance values exceeding

0.15 at 3 h were considered

mean of the absorbance

values of five negative control

3-h incubation

ofthe substrate.

at RT. Identification

the most intense color. All Specimen

positive.

Viruses were

wells with absor-

For a test to be valid, the

wells could not exceed 0.15 after

of a virus was considered

valid only if the P/N

ratio of that virus was 2-fold higher than those of all other serotypes. (P is optical density of virus specimen well, and N is the mean of optical densities of negative control wells at 405 nm.) Monoclonal antibodies All hybridomas which produced

the monoclonal

antibodies

used for sensitization

were originally isolated by Dr. Mary Kay Gentry of the Walter Reed Army Insitute Research (WRAIR), Washington, D.C. The anti-DEN 2 (3H5) and anti-DEN

of 4

(1H 10) monoclonal antibodies were described by Gentry et al. (1982) and Henchal et al. (1982) and used at 1 : 400 dilution. The anti-DEN 1 (8C2) and anti-DEN 3 (9El) monoclonal antibodies were selected on the basis of studies to characterize anti-DEN monoclonal antibodies (Dr. D.S. Burke of the WRAIR, unpubl. data). They were prepared in ascitic fluid and used at 1 : 200 and 1 : 400 dilutions, respectively. Sensitivity and specificity tests For sensitivity testing, 2-fold dilutions of the 4 DEN prototype viruses were prepared in PBS-O. 1% T and a checkerboard titration was set up using plates sensitized with serial 2-fold dilutions of homologous monoclonal antibodies. The 4 DEN prototype viruses, the 17-D strain of YF virus, the 5-3 attenuated

strain of Japanese

(JE) virus, and Ross River (RR) virus were used for specificity

testing.

encephalitis DEN viruses

were titrated by mosquito inoculation (Rosen and Gubler, 1974) or by infectivity test of TRA-284SF cell cultures using the IFA technique of Gubler et al. (1984). Virus titers

were calculated

according

to the method

of Reed and Muench

(1938) and

expressed as 50% mosquito infectious dose (MIDSo) per 100 ul for DEN or 50% tissue culture infectious dose (TCIDS,,) per 100 ul for the non-DEN viruses. Shelf life of presensitized plates Plates were sensitized with monoclonal antibodies as described above and air-dried. They were stored at -15°C for up to 4 mth, at 4°C for up to 3 mth, and at 36”Cfor up to 2 wk. The plates were tested at various intervals after sensitization for reactivity with the 4 prototype

DEN viruses

preserved

at -65°C.

Effects of DEN virus storage temperature on reactivity The prototype DEN viruses were stored at RT for 0.5, 1,2, and 3 mth and at -15°C for 6 mth and 1 yr. All tests with stored viruses were done using freshly sensitized plates.

96

RESULTS

Specificity The A&-ELISA

was highly specific for DEN virus identification

by P/N ratios in Table

1. With most viruses,

identification

as demonstrated

was unequivocal

because

only one serotype of monoclonal antibody reacted with each virus. All viruses were easily identified spectrophotometrically 1 h after substrate reaction. JE, YF, and RR viruses did not react significantly with any of the monoclonal antibodies. Sensitivity The 8C2 DEN 1 monoclonal antibody, at 1 : 200 and 1 : 400 dilutions, had a sensitivity limit of 1.5 X lo5 (Fig. 1). Higher dilutions of the monoclonal antibody required

proportionally

general,

color developed

higher

concentrations

of virus

more slowly with prototype

for a positive

reaction.

In

DEN 1 than it did with the other

3 prototype viruses. Color was clearly visible 1 h after the addition of the substrate at a virus concentration of 9.6 X 10’ MIDS,, but not at a concentration of9.6 X lo6 MID,,. A 1 : 400 dilution of the DEN 2 monoclonal antibody (3HS) was selected for tests because at 1 : 200 dilution, excessive variation in absorbance was observed with virus dilutions ranging from 3.8 X lo4 to 6 X lo5 MID,,. At 1 : 400, the sensitivity limit was 9 X lo5 MID,,. Color often developed within 30 min but its intensity was weak with low virus concentrations. Color intensity was stronger with increasedvirus concentrations of 4.8 X lo6 to 2.4 X 10’ MID,,

at 1 h incubation.

The sensitivity limit of the DEN 3 monoclonal antibody at a 1 : 400 dilution was among the lowest at 1.25 X lo3 MID,,. With higher virus concentrations of 2 X lo6 MID,,

and 2 X 10’ MID,,,

TABLE

I

Specificity

of antigen

Virus

capture

ELISA

Monoclonal DEN

DEN

clearly visible

1 (Hawaii)

DEN 2 (NC ‘C’)

for dengue

color developed

(DEN)

within

30 min.

virus identification

antibody

l-8C2

DEN 2-3H5

DEN 4-1HlO

DEN 3-9EI

9.0a

1.3

1.1

1.5

1.3

5.4

0.7

0.6

DEN 3 (H-87)

1.0

G?

10.0

0.7

DEN 4 (H-241)

0.7

0.9

.6

9.4

JEb (5-3) YFC (17D)

1.5

1.2

1.3

1.3

1.7

1.2

1.4

Ross River

1.5

1.3

1.5

1.9

* P/N: Ratio of absorbance

of specimen

OD 405 nm. h Japanese

encephalitis

’ Yellow fever virus.

wrus.

well (P) and the mean of absorbance

1.4

valuesof

negative wells(N

97

DILUTIONS OF MONOCLONAL ANTIBODY -o-

AMOUNT

Fig.

1.Titration

OF

VIRUS

I:200

(MID50/100,u(/WELL)

of dengue (DEN)

The DEN 4 monoclonal

1(Hawaii) using anti-DEN

antibody

antibody(8C2)

1 : 200 and 1 : 400 dilutions

using

limits of 7.2 X lo4 and 9.6 X lo4 MID,,, incubation.

1 monoclonal

respectively.

by A&-ELISA.

had sensitivity

Color became visible after 1 h of

Identification of viruses grown in cell culture In contrast to the prototype viruses, some of the cell culture-passaged

nonprototype

strains, in particular DEN 1 and DEN 3, could not be identified spectrophotometrically until after 3 h incubation. Incubation longer than 3 h did not enhance identification spectrophotometrically,

but rather increased

the absorbance

in negative control wells.

However, prolonged incubation enhanced visual identification and a 6-h incubation was therefore selected for this method. Geographic origins of the DEN virus strains tested

are shown

previously

in Table

identified

2. It should

by CF, monoclonal

be noted antibody

that all of these viruses IFA and/or

had been

PRNT.

DEN 1 Except for 2 Caribbean isolates, most strains of DEN 1 did not develop visible color in 1 h, necessitating longer incubation. Out of 21 strains tested, 3 (1 each from Fiji, Puerto Rico, and St. Martin) could not be identified (Table 2). Most of the identified strains had titers exceeding 2.4 X lo4 MID,,. All 3 unidentified strains had titers below the sensitivity limit (1.5 X lo5 MID,,) established for the prototype. DEN 2 Out of 29 DEN 2 strains

tested, 26 were readily identified

(Table

2). Twenty-four

98

TABLE

2

Identification Serotype

DEN

1

of dengue

(DEN)

Geographic

strains

area

grown

in cell culture

by A&-ELISA

No. strains

No. strains

identified

not identified

Asia India

0

Sri Lanka

0

Pacific Fiji

0

1

Caribbean Jamaica

0

Dominica Puerto

0

Rico

1

Mexico

0

St. Martin

1

Trinidad

0

South America Brazil Colombia Total DEN 2

3

0

-1

-0

18

3

Asia India

0

Indonesia

1

Malaysia

0

Sri Lanka

0

Pacific Fiji

2

0

Tahiti

1

0

Trinidad

1

0

Jamaica

2

1

Mexico

1

0

Caribbean

Puerto

Rico

Honduras U.S. Virgin Islands

1

1

0

- 1

-0

26

3

Indonesia

7

0

Malaysia

3

0

Sri Lanka

5

1

Thailand

3

0

0

1

Total DEN 3

8

Asia

Pacific Tahiti

99

TABLE

2 (continued)

Serotype

Geographic

area

No. strains

No. strains

identified

not identified

Caribbean Puerto

Rico

Total DEN 4

3

4

21

6

1

0

Asia Indonesia Caribbean Mexico

2

St. Barthelemy

1 1

Dominica Puerto

13

Rico

Dominican

1

Republic

South America Brazil

0

3 -

-

1

22

Total

strains developed color in 1 h and could be visually identified, while the other 2 strains could be identified visually only after 3-6 h incubation. The 3 strains that could not be identified were from Indonesia, Jamaica, and Puerto Rico. Virus titersofthe majority of the identified viruses ranged between IO3 and lo4 MID,,, which were lower than the sensitivity

limit (9 X lo5 MID,,)

titers below the sensitivity

of the prototype

virus. All unidentified

strains

had

limit.

DEN3 Out of 27 DEN 3 strains tested, 21 were identified visually

(Table 2). Most of the 19 southeast

both spectrophotometrically

and

Asian strains tested had virus titers ranging

to or higher than the from 1.2 X lo3 to 4 X IO5 MID,,, which were comparable sensitivity limit of the prototype DEN 3 (1.2 X 103MID,,). Color usually developed within 3 h for the Asian strains, while it generally developed more slowly with Puerto Rican strains. Of the 6 unidentified 1 from Sri Lanka. All unidentified

viruses, 4 were from Puerto Rico, 1 from Tahiti and viruses had titers below the sensitivity limit of the

prototype virus. Of the 3 Puerto Rican strains that were correctly identified, 2 had virus concentrations higher than the prototype virus sensitivity limit. One of these, PR-6, was identified only when high concentrations of virus were used in plates sensitized with twice the concentration of 9El monoclonal antibody as was optimal for the prototype virus. Under the above conditions, the sensitivity limit for the PR-6 strain was 1.8 X lo5 MIDSo.

100

DEN 4 Out of 23 DEN 4 strains were identified between

visually

tested, 22 were readily identified

within

1 h after addition

1.5 X lo3 and 2 X lo5 MID,,.

prototype

sensitivity

(Table

of the substrate,

The unidentified

2). Most viruses

with titers ranging

strain had a titer below the

limit (9.6 X lo4 MID,,).

Shelf life of presensitized plates The shelf life of plates presensitized 4°C and -lS’C,

respectively.

tized plates was evident

with monoclonal

The deterioration

after storage

antibodies

of monoclonal

was 2 and 4 mth at

antibodies

in presensi-

for 3 mth at 4°C.

Storage temperature of dengue viruses Prototype viruses were still correctly identified

after storage at -15’C for 1 yr and at

RT for 1 mth. The viruses incubated at RT for 1 mth were inactivated, but those stored at -15°C were still infectious. Incubation at RTfor longer than 1 mth prevented correct identification. DISCUSSION

For choosing a proper sensitivity but also affinity

sensitizing antibody in ELISA, not only specificity and constant must be taken into consideration (Yolken, 1982).

Part et al. (1979) used human anti-DEN sera and hyperimmune mouse ascitic fluids (HIMAFs) for DEN EIA. However, Monath and Nystrom (1984) found that monoclonal antibody was more sensitive than human IgM for YF antigen capture from human sera with high virus titers, whereas human IgM capture antibody was more sensitive with low-titered viremic sera. Decreased sensitivity in EIA for influenza and herpes

viruses

sites of large

has been explained protein

antigens

by the fact that ‘antibodies to produce

the high-affinity

must react at different binding

required

for

successful immunoassay systems’ (Yolken, 1982). This could explain the slow color development of many nonprototype DEN strains observed in our tests. On the other hand, monoclonal antibodies provide more specific reaction than polyclonal

antibodies.

each of 4 DEN

In our preliminary

serotypes

for sensitizing

tests we used HIMAFs plates,

prepared

but the nonspecific

against

reaction

that

developed with DEN 3 and DEN 4 HIMAFs prevented accurate identification with AgC-ELISA (data not shown). The superior specificity of monoclonal antibodies to polyclonal antibodies has been previously documented in other viral EIAs (Beards et al., 1984; Goldstein et al., 1983; Nilheden et al., 1983; Portetelle et al., 1983). However, not all type-specific monoclonal antibodies were useful for AgC-ELISA identification of DEN viruses. In the preliminary study (not shown) we evaluated the 15F3 DEN 1 and 5D4 DEN 3 monoclonal antibodies, successfully used in immunofluorescent antibody test for DEN identification (Gubler et al., 1984), but found them nonreactive in the AgC-ELISA.

101

The difference

in sensitivity

2 and DEN 4 monoclonal ly producing visual viruses

among

antibodies

color in 1 h, while incubation

identification

with DEN

prototype

virus. However,

antibodies

was quite evident.

for more than 1 h was generally

1 and DEN

that could not be identified

respective

monoclonal

reacted much faster and more strongly, 3 monoclonal

antibodies.

required for Most of the

had virus titers below the sensitivity there were a number

DEN

frequent-

limit of the

of viruses that were readily

identified by AgC-ELISA even though the virus titers were lower than the sensitivity limit of the homologous prototype. One strain of DEN I, 9 strains of DEN 2,2 strains of DEN 3 and 2 strains

of DEN 4 fell into this category.

1 strain of DEN 3

By contrast,

from Puerto Rico could not be identified even though the virus titer was comparable to the sensitivity limit of the prototype, and another DEN 3, also from Puerto Rico, was identified only after the virus titer had been increased to lOO-fold higher than the prototype sensitivity limit. Thus, although the sensitivity limits established for prototype viruses may be useful as a guide for correct identification, they alone cannot be used to evaluate the AgC-ELISA for DEN virus identification because of the variation in sensitivity limits. These results suggest possible strain variation among DEN viruses in their reactivity to monoclonal antibodies and/or in their production of noninfectious viral components. It is of interest to note that another was from Tahiti. This virus, and the virus titer was below others (Russell and McCown, antigenically similar to each ic strain variation observed However, all DEN viruses antibody immunofluorescent Variation AgC-ELISA,

in sensitivity

noted above were readily identified test. has been observed

the sensitivity

With Coxsackie

of the DEN 3 viruses that could not be identified

like most of the Puerto Rican viruses, replicated poorly the sensitivity limit of the prototype. It has been shown by 1972) that the Puerto Rico and Tahiti DEN 3 viruses are other, but different from Asian strains. Thus, the antigenby neutralization test was also demonstrated by ELISA. with the monoclonal

with other viruses.

limit was 2 X IO3 TCIDJ200

In the polio virus

ul (Payment

et al., 1982).

and echo viruses, the limits ranged from lO*,i* ‘cytopathogenic

to 106.t4 plaque forming

units’

units (PFU) per well (Deng and Cliver, 1984). In herpes virus

AgC-ELISA, it was about 90 PFU or 6 X lo3 physical particles per 50 ul (Alder-Stortz, et al., 1983). Similarly, the sensitivity limit of Coxsackie B virus was lo3 TCID,,/ml (Yolken

and Torsch,

1980). In YF AgC-ELISA,

YF virus was 0.007-0.0029

g viral protein

the maximum

or 103.3-3.6 PFU/SO

sensitivity

with purified

ul (Monath

and Nys-

trom, 1984). In our tests we expressed sensitivity limits in MID,,, since DEN virus assay by mosquito inoculation is recognized to be the most sensitive technique (Rosen and Gubler, 1974). In our AgC-ELISA, most viruses were readily identified spectrophotometrically in 3 h. If the incubation period was extended to 6 h, most of the same viruses could also be identified visually. Although visual identification has an inherent drawback in that it is subject to the perception of readers, the fact that color development monotypic reduced the probability of erroneous interpretation.

was generally

102

Various

techniques

din ELISA

(1984), and Inouye disrupt

have been applied to improve

is reported

rotavirus

to amplify the reaction

et al. (1984), used EDTA

particles

sensitizing

antibodies.

time and improve

Leister,

Constant

agitation

sensitivity

1981). In our study, neither

Biotin-avi-

et al., 1983). Beards et al.

and chaotropic

to expose viral antigen

incubation

the results of ELISA.

(Alder-Stortz

agent,

respectively,

which may be more reactive

at 36°C for 10 min was reported of ELISA (Portetelle

EDTA

treatment

to

to the

to reduce

et al., 1983; Yolken and

of DEN

1 strains

nor constant

agitation improved results significantly (data not shown). The latter was carried out at 4°C overnight, at 36°C for 3 h during sensitization of the solid phase and at 36°C during virus antigen reaction. The AgC-ELISA we developed is an economic and specific technique. Thus, the majority

of newly isolated

expensive

ELISA

reader.

DEN viruses Furthermore,

can be identified

visually

since presensitizeds

without

use of an

plates can be held in a

regular freezer (-15’C) for at least 4 m without loss of reactivity of capture antibodies identification can be accomplished within a day. Finally, because viruses can still be identified after 1 yr storage at -15°C our method may be useful in those laboratories that do not have low temperature freezers (-65°C) necessary for virus preservation. A drawback of the technique is that large amounts of viral antigen are necessary for identification because of high sensitivity limits of the monoclonal antibodies used. Although most viruses were readily identified, there were some that could not be identified even though virus titers were high. ACKNOWLEDGEMENTS

We thank

Drs. T. Monath

and J. Roehrig

of the Centers

Collins, Colorado, for advice and provision DEN 2 and DEN 4 monoclonal antibodies; Walter Reed sion of DEN M. Velez, E. contribution Health

Army 1 and Vergne of the

Grant

for Disease Control

at Ft.

of alkaline-phosphatase conjugate, and Drs. M.K. Gentry and D.S. Burke of the

Institute of Research, Washington, D.C., for selection and proviDEN 3 monoclonal antibodies. Technical assistance of A. Oliver, and N.A. Colon of the San Juan Laboratories is appreciated. The third author (N.S.W.) was supported by the National Institute of

NIGSH-GM-029

19-03.

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