Virus enzyme-linked cell immunoassay (VELCIA): Detection and titration of rotavirus antigen and demonstration of rotavirus neutralizing and total antibodies

Journal

ofVirological

Methods,

135

10 (1985) 135-144

Elsevier JVM 00366

VIRUS

ENZYME-LINKED

AND TITRATION ROTAVIRUS

J. GROM* I.N.R.A.,

(Accepted

NEUTRALIZING

Pathologic

Porcine.

16 October

Virus enzyme-linked

F-37380

ANTIGEN

AND TOTAL

(VELCIA):

DETECTION

AND DEMONSTRATION

OF

ANTIBODIES

cell immunoassay

material

24 h. Porcine

within

Nouzilly.

porcine

antigen

for detection

and titration

can be detected

can be titrated

of rotavirus

of the cell culture system for virus isolation

rotavirus

(VELCIA)

rotavirus

OSU strain

for the demonstration reading

France

1984)

Wild-type

metrical

IMMUNOASSAY

and S. BERNARD

been developed. introduced

CELL

OF ROTAVIRUS

neutralizing

are combined

higher

of rotavirus

and titrated

than

directly

10.“. The method

and total antibodies.

In VELCIA

with enzyme immunodetection

antigen

has

from the fecal has also been the advantages

and spectrophoto-

of the test.

VELCIA

detection

of antigen

neutralizing

titration

antibodies

total antibodies

INTRODUCTION

Rotaviruses have been associated with diarrhea animals of various species (Flewett and Woode,

in children and a number of young 1978). Many techniques have been

developed for rotaviral antigen detection from specimens and fecal material: electron microscopy and immunoelectron microscopy (Bishop et al., 1974; Grom et al., 1980); immunofluorescent staining of sections, smears or fixed inoculated cell cultures (Woode et al., 1976; Theil et al., 1977); counter immunoelectrophoresis (CIEP) (Middleton et al., 1975); radioimmunoassay (RIA) (Middleton et al., 1977); enzymelinked immunosorbent assay (ELISA) (Scherrer and Bernard, 1977) and solid phase aggregation

of coated

The test described

erythrocytes in this paper

(SPACE)

(Bradburne

has several features

et al., 1979). in common

with immunope-

roxidase-labelled technique (Chasey, 1980) for the detection of rotaviral antigen. We confirmed the presence of specific bound enzymes to antigen in fixed cell sheet on microtiter plates by revealing antirotaperoxidase activity with soluble substrates.

* To whom correspondence

Biotehniska

fakulteta,

0166.0934/85/$03.30

should be addressed

Univerza

E. Kardelja

(present address):

v Ljubljani.

s 1985 Elsevier Sctence Publishers

Joie

Grom,

61000 Ljublja’na, B.V. (Biomedical

VTOZD

za veterinarstvo,

Gerbiceva

60, Yugoslavia.

Division)

Rotavirus

neutralizing

tion test (Thouless

antibodies

1980). The types of rotaviruses cultures

are usually detected

et al., 1977) or neutralizing

were determined

(Sato et al., 1982) or microplates

We described demonstration MATERIAL

antibody

by a neutralization

(Gaul

the use of virus enzyme-linked of neutralizing

by fluorescent

focus reduc-

plaque assay(Vautherot

et al.,

test in tube cell

et al., 1982). cell immunoassay

and total antibodies

against

(VELCIA)

for the

rotavirus.

AND METHODS

Sera Sera were collected from 5-6 mth-old Sera were heat-inactivated essential medium (MEM).

(56°C

pigs from large farms in Slovenia,

30 min) and diluted

Yugoslavia.

11100 in Eagle’s

minimal

Viruses Rotavirus samples

OSU strain

(Dr.

Bohl) and wild-type

porcine

rotaviruses

from fecal

were used.

Cell culture Confluent sheets of fetal rhesus monkey MA- 104 cells were grown at 37°C in plastic flasks (Corning) in MEM (Eurobio) supplemented with 10% fetal calf serum (Gibco), 100 U/ml penicillin and 100 ug/ml of streptomycin. For VELCIA, cells were trypsinized (trypsin + versen) and resuspended containing IO6 cells/ml. To each well of polystyrene 96well microplates 3042), 50 ul of ceils suspensions monolayer

became

were added and incubated

in MEM (Falcon-

for 48-72 h when the cell

confluent.

infectivity assay for non-adapted rotav~r~~ses From fecal samples 20% suspensions were prepared in phosphate-buffered saline (PBS) and clarified at 3,000 r.p.m./30 min. Supernatant fluids of samples were inoculated

in 50 ul amounts

into 4 wells of microplate

containing

50 @‘well MEM

supplemented with 30 pglml pancreatin (Sigma). After an activation for 90 min at 37’C, the samples were transferred with a multichannel pipette (50 ~1) into a microplate containing a confluent monolayer of MA-104 cells rinsed 3 times with MEM. After adsorption of 60 min at room temperature, the samples were discarded and the plates rinsed again 3 times with MEM, maintained in 100 $/well of MEM and incubated for an additional 18-20 h. Positive and negative controls were included in every microtiter plate. Titration of rotavirus OSiJ strain Serial IO-fold dilutions of viruses were prepared in MEM containing 30 ug/ml of pancreatin. After an activation of 60 min, each dilution was inoculated into 16 wells of

137

a microtiter

plate with a confluent

and incubated

sheet of MA-104 cells rinsed 3 times with MEM,

for 48 h at 37°C.

Ro~a~~~rus antigen detection by the VELCIA ~echnjque Fixation of the ce!l sheet. After an incubation of the infectivity assay or virus titration the cell sheet on microtiter plates was fixed by the use of precooled (-20°C) 85% acetone in distilled water, sealed with tape and stored for a maximum of 24 hat -20°C as longer storage in acetone can reduce the transparency of the microplate. Acetone was discarded

and microplates

were air-dried.

Fixed dried microplates

can be used at

once or stored sealed with tape at -20°C (tested conservation time 10 days). Microplates were washed before and between each incubation of VELCIA 3-5 times with saline containing

0.05% Tween

20.

Specific ~neubar~ons. Microplates containing 50 pi/well porcine antirotaperoxidase conjugate (Nakane and Kawaoi, 1974), diluted l/200, were incubated for 2 hat 37°C. Subsequently we added 100 ul/well of ABTS substrate (2,2-azino-di-3-ethylbenzothiazoline sulfonic acid, Boehringer). The reaction was stopped after 1 h by transferring 80 ul/well of substrate solutions to a new microplate containing 20 pb’well of 10% sodium dodecyl sulphate (SDS). Reactions were read at 405 nm on a spectrophotometer with a microplate reader (Vernon). Rotavirus antigen detection by other techniques Immunopero~~dase assay-~nso~~b~eprodigy. The microplate on which the peroxidase activity was revealed in VELCIA was washed 3 times with tap water and specific rotavirus-peroxidase reactions on the cell sheet were labelled by adding 3,3’-diaminobenzidine

(DAB) (diluted

10 mg/ml

rotaviral

positive foci were counted

ELZSA.

ELISA for rotavirus

ed in details

by Scherrer

in Tris buffer and 0.01% H,O,). and the results compared

antigen

and Bernard

Immunofluorescent lube&g.

detection

The infectivity

with those ofVELCIA.

from fecal samples

(1977) and Bernard

Brown labelled

has beendescrib-

et al. (1984).

assay and fixation

of the cells on micro-

plates were the same as for VELCIA. A 50 ~1 of antirotafluorescent conjugate (swine serum against OSU strain, diluted l/IO) was added to each well, incubated for 1 h at 37°C and washed 3 times for 10 min with tap water. The rotavirus fluorescent foci were examined on inverted microplates using fluorescent microscope (Leitz). CZEP. CIEP was performed Middleton et al. (1977).

by the modified

method

of Grom

et al. (1982) and

13X

Rotavirus

antibodies

detection

by VELCIA

technique

VELCIA total antibodies (VELCIA-total). Microplates with a confluent monolayer of MA-104 cells were rinsed 3 times with MEM and inoculated with 1000 TCID,,/SO ul of activated

(pancreatin

11 rows of microplates.

30 ul/ml MEM, 1 h of activation)

rotavirus

One row (8 wells) was not inoculated

OSU strain into

and was used for cell

control. After an adsorption for 1 h, the virus suspension was discarded and microplates rinsed again with MEM, maintained with 100 PI/well of MEM and incubated for 24 h at 37°C. The cells on microplates described previously. Total anti-rotavirus

antibodies

were fixed with cold 85% acetone

were determined

on VELCIA

microplates

as

as fol-

lows: the dilutions of sera (l/1000 and l/2000) in PBS (0.05% Tween + 0.1% HSA) were inoculated into 4 wells (50 pi/well) and incubated for 3 h at 37”C, 100 $/well of anti-swine total immunoglobulin conjugate of ABTS were added after 1 h of incubations with multichannel to stop the reaction. microplate

reader

(incubation 2 h at 37°C) and 100 pi/well at 37”C, and 80 ul/well were transferred

pipette to a new microplates

with 20 ul/well

of 10% SDS solutions

The results were read by the use of a spectrophotometer

with a

(Vernon).

VELCIA neutralization antibodies (VELCIA neutral). The dilutions assayed (l/100, l/1000, l/2000) were prepared with MEM in parallel

of sera to be in 4 wells of a

microplate. Dilutions of sera were mixed in an equal volume (50 pi + 50 ~1) with pancreatin preactivated (30 ug/ml, activated for 1 h at 37°C) suspensions of OSU rotavirus containing 500 TCID,,. Virus-sample mixtures were incubated for 2 h at 37°C and then transferred with a multi-channel pipette (100 ul) into a microplate containing adsorption

a confluent monolayer of MA-104 cells rinsed 3 times with MEM. After an for 1 h at room temperature, samples were discarded and plates rinsed

again twice with MEM, maintained For the demonstration -20°C) were incubated

with 100 ul/well

of MEM and incubated

for 48 h.

of neutralizing antibodies fixed microplates (85% acetone, successively with antirotaperoxidase conjugate (2 h at 37°C)

and ABTS (1 h at 37°C). Transfer,

stopping

the same as those described

for VELCIA-total.

above

and reading

of substrate

solutions

were

Evaluation of resufts. The mean of optical density (OD) vaules (4 wells or 16 wells) of a rotavirus infected cell sheet were compared with the mean of OD values obtained from a non-infected cell sheet (minimal 6 wells). The results were also compared with positive and negative control samples on each plate. Only values statistically (Student’s t-test) significantly higher than in non-infected cells were counted as positive.

139

RESULTS

Rotavirus

antigen

Rotavirus

infectivity

assay.

Pancreatin

activated

wild-type

porcine

rotavirus

can be

detected within 24 h on cell culture using VELCIA. Reading by spectrophotometer permits distinguishing positive from negative samples. Thirty-seven samples collected from 20 pigs were tested. We have found a good correlation between results of two VELCIAs P
A good correlation

VELCIA/VELCIA VELCIA/ELISA VELCIA/CIEP

Y = 0.664 r = 0.376

P
r = 0.716

P
ELISA/CIEP

r = 0.679

P
Titration of rotavirus. (OD

Wild-type

porcine

rotavirus

between

can be detected directly from the

405nm)

1 .I_

0.9_

0.7_

O.S_ L

I

1

0.3 Fig. 1. Correlation

!

I

I

1

between

two VELCIA

J (OD 405nm)

1

1.1

0.7 tests for rotawrus

the tests was

antigen detection

140

feces in dilutions yielded

higher than

titers greater

dilutions

this dilution control

than

higher than

2 X 1tY3. After the second

10w4. Reference

10m8(50 $/well).

was still statistically

non-infected

porcine

OSU strain

VELCZA-total.

Total antibodies

total antibodies

cells

could be detected

in at

higher than the mean of OD values of

(1.5 wells) (Fig.

2). The virus

cytopathology was three log,, dilutions lower (10e5, 100 $/well) tions lower as determined by immunofluorescence.

Although

on MA-104

The mean of OD values (16 wells) obtained

significantly

cell sheets

passage

against rotavirus

can be indirectly

titer examined

and also two dilu-

can be demonstrated

determined

by

by VELCIA.

using the antirotavirus

conju-

gate, the sensitivity of the test is better when using anti-swine total immunoglobulin conjugate. Comparison of 35 sera in two VELCIA-totals gave a good correlation (r= 0.666; P = 0.0014) (Fig. 3).

VELCIA-neutral.

The test clearly discriminates

between rotavirus

positive and nega-

tive sera in the presence of neutralizing antibodies. Thirty-seven sera tested twice in VELCIA-neutral showed a good correlation: r = 0.733; ~
VELCIA and other tests. ELISA, number

COD

all VELCIA-neutral of ELISA positive

When

VELCIA-neutral

was compared

positive sera were also ELISA positive. sera were negative in the VELCIA-neutral

with

indirect

In contrast, test.

a

405nm)

1

1

I

Dilution Fig. 2. Titration

1

I

-6

-4

of porcine

-8 (Log

rotavims

IO) OSU strain by VELCIA:

(0) The mean of optical density (OD) for the

viral dilutions

(16 values). (A) The mean of OD of the blank values (15 values). The abscissa indicates the

viral dilutions

(loe,~,).

141

COD

405nm)

1 _

0.7,

0.4, I

L

1

1

COD Fig. 3. Correlation

1

0.3

0

between

I

1

0.6

405nm) two VELCIA-total.

405nm)

COD

0.6_

0.3, I

0.2

1

I

1

COD Fig. 4. Correlation

between

I

0.6

1

405nm)

two VELCIA-neutral.

I

1

0.9

J

142

In parallel

3 1 sera were examined

by three tests: indirect

ELISA,

VELCIA-neutral

and VELCIA-total. n

r

P

VELCIA-neutral/VELCIA-neutral

?-?

0.732

VELCIA-neutral/VELCIA-total

31 31 35

0.035 0.132 0.666

<0.0001 >0.05* >0.05”

31

0.511

VELCIA-neutral/ELISA VELCIA-totai/VELCIA-total VELCIA-tota~/ELISA *No significant correlation.


There was no correlation between VELCIA-neutral and VELCIA-total and between indirect ELISA and VELCIA-neutral test. Non-neutralizing components ofsera could produce discrepancies between found between the results of indirect

the results of the tests. Good correlations were ELISA and VELCIA-total as both techniques

detect the same type of antibodies, DISCUSSION

This study was made to investigate the use of enzyme immunoassay for the detection of rotavirus antigen in cell monolayers on microtiter plates. The cultivation of the rotavirus on the cells was not easy, but it was observed that wild-type rotavirus readily infects cells using trypsin or pancreatin pretreatment and rotaviral antigen can be detected on a cell sheet by immunofluorescence (Theil et al., 1977; Bridger and Brown,

1981) or immunoperoxidase

labelling

(Chasey,

1980). VELCIA

has been

developed as a continuation of this generation of tests on cell cultures, by improving the sensitivity and objectivity. Enzyme immunoassays have been developed for screening monoclonal antibodies against

cell surface

antigens

using fixed cells on microplates

(Cobbold

and Wald-

mann, 1981: Feit et al., 1983), but few authors have used enzyme-conjugated antibodies to detect inner antigen on the fixed cell sheet on microtiter plates (Saunders, 1977). Only a limited number of porcine rotaviruses can be serially passaged with cytopathology (Theil et al., 1977; Bohl, 1979; Fukusho et al., 1981) and exact titers are difficult to obtain. For the titration of adapted rotavirus strains the plaque technique is used, which is inconvenient and time-consuming (Bohl et al., 1980; Kalica et al., 1983). With VELCIA we were able to titrate OSU strain of porcine rotavirus in dilutions higher than 10Y8 (50 pi/well) and also directly from the feces wild-type porcine rotavirus in dilutions higher than 10e3 (50 pillwell). This is not possible using the plaque technique. By the use of the spectrophotometer reliable results are obtained in 48 h. Using cell cultures. specific antibodies may be demonstrated by the neutralization

143

test. Infectivity the complete zation

of rotavirus

is also located

antibodies

for cell culture

particle (Bridger and Woode, was difficult

1982); therefore

is associated

with the outer capsid layer of

1976) and antigen

associated

at this site (Bridger,

1978). The detection

as the cytopathogenic

effect of rotavirus

the fluorescent

or plaque

technique

with neutraliof neutralizing

is weak (Gaul et al.,

was applied.

Enzyme

labelling

was proposed to facilitate visual reading of weak virus cytopathology (Anderson and Rowe, 1982) but there were few attempts at instrumental reading (Saunders, 1977). In the present study enzyme immunoassay detection. VELCIA permits demonstration antibodies

using the same cell culture

VELCIA was developed for antibody and titration of neutralizing and total

system as used for virus isolation

and titration.

The sensitivity of VELCIA-neutral and VELCIA-total was good, even a considering problem in the level of the background. The results of sera tested concomitantly with VELCIA-neutral, VELCIA-total and indirect ELISA showed that the results were not comparable, as these techniques detect different types of antibodies. Some of the sera positive for total antibodies (detected by VELCIA-total or indirect ELISA) were negative in VELCIA-neutral. These results were obtained with one virus strain (OSU), which does not exclude the possibility of a neutralization against other rotavirus serotypes. The discrepancy in the results shows the limited value of total antibody

detection

for epidemiological

studies

of rotavirus

infections.

In VELCIA the advantages of cell culture, enzyme immunoassay and spectrophotometrical reading are combined for the isolation and titration of rotavirus and also for the demonstration and titration of neutralizing and total antibodies. The test is easy to perform not only on microplates, also conventional glass tissue culture tubes can be used, but it will remain reserved for laboratories with tissue culture facilities. There is no doubt that this technique can be adapted for antigen detection of viruses with a weak or without cytopathogenic effect.

and antibody

ACKNOWLEDGEMENTS

We thank Dr. J.M. Aynaud excellent technical assistance.

for his helpful advice and Mrs. I. Lantier for providing

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