Use of immunosorbent electron microscopy for detection of rota- and hepatitis a virus in sucrose solutions

Journal of Virological Methods, 12 (1985) 161-167

161

Elsevier JVM 00446

USE OF IMMUNOSORBENT

ELECTRON

OF ROTA- AND HEPATITIS

ELISABETH

KJELDSBERG

MICROSCOPY

A VIRUS IN SUCROSE

and JENS CHRISTIAN

FOR DETECTION

SOLUTIONS

SIEBKE

Virological Department, National Institute of Public Healih, Geitmyrsveien 75, 0462 Oslo 4, Norway (Accepted

30 July

1985)

Immunosorbent concentrations achieved

electron

microscopy

was used to demonstrate

after 18,42 and 66 h of incubation.

after 18 h incubation

and nearly

rotavirus

in solutions

About 50% of adsorption

of varying

of virus particles

100% after 42 h when compared

to trapping

sucrose

to the grid was

of virus from sucrose

free solutions. Hepatitis

A virus

immunosorbent

was purified

electron

dioimmunoassay.

The sensitivities

essentially

and considerably

electron

similar

in a lo-30%

microscopy,

direct

sucrose

electron

of immunosorbent greater

than

gradient

microscopy, electron

and each fraction immune

microscopy

direct electron

electron

was examined microscopy

and radioimmunoassay

microscopy

by

and ra-

and conventional

were immune

microscopy.

immunosorbent

electron

microscopy

rotavirus

hepatitis

A virus

sucrose

solutions

INTRODUCTION

Direct

electron

microscopy

(EM) and conventional

(IEM) have been used commonly (Feinstone

electron

microscopy

in sucrose or salt gradients

et al., 1974; Bradley et al., 1975; Flewett and Boxall, 1976). The presence of

low molecular it difficult

immune

for locating virus particles

material

to obtain

such as sucrose or salt in high concentrations,

good results by the negative

staining

technique.

however, makes Procedures

used

earlier for removing the sucrose prior to electron microscopy such as dialysis, pelleting and resuspension of material or evaporation and washing of the specimen on the grid are cumbersome, unreliable and of low sensitivity. Immunosorbent

electron

microscopy

(ISEM)

is widely used for detection

of virus

particles in clinical specimens and cell culture material as reviewed by Katz and Kohn (1984). It is a highly sensitive technique which allows thorough washing of the specimen and thereby removal of contaminating material without loss of virus particles. Plant viruses have been demonstrated in diluted or undiluted samples taken from sucrose gradients by this technique using specific antibody coating of virus particles adsorbed to the grid (Milne and Luisoni, 1975) or by coating of the grid with specific antibody prior to virus adsorption (Lesemann et al., 1980). In this report we describe 0166-0934/85/$03.30

0 1985 Elsevier

Science Publishers

B.V. (Biomedical

Division)

162

the application

of a protein

high concentrations

A - antibody

of sucrose

and radioimmunoassay

coating ISEM for rotavirus

and a comparison

(RIA) for detection

of ISEM,

of hepatitis

suspensions

EM, conventional

with IEM

A virus in sucrose gradient

fractions. MATERIALS

AND

Virus Human

METHODS

rotavirus

was extracted

from faeces as described

earlier (Kjeldsberg,

1977)

in Hanks’ salt solution supplemented with bovine serum albumin and antibiotics. Sucrose in 0.1 M phosphate buffer, pH 7, was added to the virus suspension to final concentrations of 10, 20 and 40%. Hepatitis A virus (HAV) was propagated in fetal rhesus monkey kidney cells (Frhk-4R). The cells were detached from the Roux bottle with glass beads, washed by centrifugation and lysed by freeze-thawing two times in the presence of 2% Triton X-100 in phosphate buffered saline. Cellular debris was removed by low speed centrifugation. The supernatant was layered on a lo-30% semi-linear gradient of sucrose in phosphate buffered saline containing 0.5% carrier bovine serum albumin and centrifuged for 3 h at 27,000 rpm in a Beckman SW 50.1 rotor. The collected fractions were stored at 4°C. Antisera Rabbit

anti-(human)

rotavirus

serum was prepared

as described

previously

(Kjelds-

berg and Mortensson-Egnund, 1982). The serum had a titre of 10,240 in fluorescent antibody test (FAT). As antiserum to hepatitis A virus, a human convalescent serum with hepatitis

A antibody

All serum dilutions

titre of 2,000 when tested by radioimmunoassay

were made in 0.1 M phosphate

buffer,

was used.

pH 7, with 0.02% sodium

azide added. Direct electron microscopy A 10 ul sample was placed on formvar carbon-coated copper grid and allowed to evaporate until nearly dry. The grid was dipped into distilled water twice, and in 2% potassium phosphotungstate (KPT), pH 6.5, once. Excess water was removed with filter paper and the grid was examined

in a JEM

1OOB electron

microscope.

Immune electron microscopy Conventional IEM was performed by mixing equal amounts (50 ~1) of virus suspension and antiserum diluted 1 : 50, incubation overnight at 4”C, the mixture was diluted three times with 0.1 M phosphate buffer and centrifuged for 1 h at 17,000 rpm in Sorvall centrifuge, SS-34 rotor. The pellet was suspended in 2% KPT, pH 6.5, and examined in the electron microscope.

163

Immunosorbent ISEM

electron

was performed

1982). Antiserum dilution dilutions

microscopy as described

against

human

earlier

rotavirus

(Kjeldsberg

and Mortensson-Egnund,

was used at a dilution

1: 2,500. Optimal

of the antiserum against hepatitis A virus was established by testing serial of the serum. The grid was pretreated with protein A, 10 pg/ml (Pharmacia),

washed with 0.1 M phosphate buffer, pH 7, coated with antiserum, washed with buffer and incubated with virus suspension. After adsorption of the virus particles the grid was washed with distilled water, negatively stained with 2% KPT and examined in the electron microscope. Radioimmunoassay Wells of Costar 2595 vinyl assay plates were coated with rabbit

IgG against human

IgM and subsequently with a 1 : 4,000 dilution of a serum from a patient with acute hepatitis A virus infection. Fractions to be tested were diluted 1 : 10 in buffered saline/carrier albumin. 50 ul samples were incubated in the wells overnight at ambient temperature. After washing, 50 ul of 1251-labelled anti-HAV (obtained from an Abbott HAVAB-M test kit) was added to each well and incubated for 4 h at 37°C. After final washing, wells were cut with a pair of scissors and counted. RESULTS

Suspensions examined

of human

rotaviruses

by immunosorbent

in 0, 10, 20 and 40% sucrose

electron

microscopy

and reaction

solutions

were

times of 18,42 and 66

h were used for adsorption of the virus particles to the grids. The results are summarized in Table 1. In the absence of sucrose, maximum adsorption of virus was achieved after reaction for 18 h and remained constant at further incubation. With sucrose in increasing concentrations, the number of trapped virus particles was reduced to about

TABLE

1

Adsorption

of rotavirus

concentration

on protein

and incubation

% Sucrose

A and antibody

electron

microscopy

grids at varying

No. of particle? 18 hb

42 hb

66 hb 104

0

95

72

10

52

85

70

20

52

91

72

40

45

74

99

a Mean count b Reaction

coated

time

on five 60 X 90 mm micrographs

time for binding

of virus.

taken

at a magnification

of X 10,000.

sucrose

164

50% at 18 h incubation. 66 h resulted electron

Prolongation

in almost

microscopy

maximum

of the reaction

adsorption

optima1 conditions

grids were coated with serial dilutions

Fig. 1. Rotavirus direct

trapped

EM. Negatively

TABLE

also. Direct

particles or none

la,b).

In order to establish prepared

of virus to 42 or

from the sucrose solutions

of the same samples showed only a few rotavirus

at all on the grids (Fig.

cell cultures

time for binding

as described

on erectron

stained

for the demonstration

of antiserum

to HAV. Hepatitis

above was used as antigen

microscopy

with 2% potassium

of HAV by ISEM,

grids in the presence phosphotungstate,

A virus from

in the test (Table

2).

of 15% sucrose by (a) ISEM and (b) pH 6.5. Bars represent

200 nm.

2

Adsorption

of hepatitis

Serum dilution

A virus on protein

1:

200

179

1:

400

257

1:

800

375

1:

1,600

374

1: 3,200

379

1: 6,400

275

1: 12,ROO

119

a Reaction

coated

grids at varying

antiserum

No. of particlesh Positive

1: 25,600

A and antibody

serum

Negative

serum

48 time for binding

b Mean count

of virus was 18 h.

on two 60 X 90 mm micrographs

taken at a magnification

of X 25,000.

dilution’

165

Dilutions

of 1: 800 to 1: 3,200 gave maximum

1: 2,000 serum dilution No virus particles Purification lo-30%

adsorption

was chosen for the examination

were seen on the grids coated

of hepatitis

sucrose gradient.

A virus propagated Each fraction

of virus

particles

of sucrose gradient

with negative in cell cultures

was examined

human

and

fractions.

serum.

was performed

in a

by RIA, ISEM, direct EM and

conventional IEM and the results are presented in Table 3. Virus particles were demonstrated in all fourteen fractions by RIA and ISEM. Conventional IEM revealed the presence of HAV in fractions 3,5,7,8 and 10, while virus was demonstrated only in fraction 8 by direct electron microscopy. Figure 2 illustrates the amount of virus antigen and virus particles in the fractions as measured by RIA and ISEM, respectively. In spite of minor deviations the shape of the graphs are almost identical and the top fractions are easily selected.

=o

:

i

2

150.

2

4:

:

2

2 3

.E ?lOO-

.3 5 zi (D

P k

x

2

-2 0

: 50. .o .L >

z”

c: -1

z

12345676

9

Fraction

Fig. 2. Demonstration 18 h). Number magnification

TABLE

of hepatitis

of virions

10

11

12

13

I

14

nr.

A virus in a lo-40%

trapped

sucrose gradient

on the grid was counted

by RIA and ISEM (incubation

on 60 X 90 mm micrographs

taken

time at a

of X 25,000.

3

Comparison microscopy

of radioimmunoassay

(RIA), immunosorbent

(IEM) and direct electron

microscopy

electron

(EM) for detecting

microscopy hepatitis

(ISEM), immune electron A virus in a lo-30%sucrose

gradient Method

Fraction

no.

1

2

3

4

5

6

7

RIA

+

+

+

+

ISEM IEM

+ _

+ -

+ +

+ -

EM

-

-

-

-

8

9

+

+

+ +

+ -

-

-

10

11

12

13

14

+

+

+

+

+

+

+

+

+ +

+ +

+ -

+ +

+ -

+ -

+ -

+ -

-

+

-

-

-

-

-

-

166

DISCUSSION

Previously gradient

used electron

fractions

EM to remove

microscopy

methods

have been unsatisfactory. sucrose

or dialysis

for detection

Extensive

washing

and centrifugation

of viruses

in sucrose

procedures

by direct

procedures

for IEM cause

dramatic loss of material and thereby a low sensitivity. Coating of grids with antibody prior to adsorption of virus would overcome this problem as the virus particles are firmly trapped on the grid by antibody without loss of virus.

and thorough

washing

may be performed

The time required for maximum adsorption of virus particles from sucrose solutions was determined as increasing viscosity of the solution has a restraining effect on diffusion

of the particles

and thereby prolongs

the reaction

time for maximum

binding

of virus. To achieve maximum adsorption of virus from solutions of high sucrose concentration 42 h incubation was necessary. If, however, the sensitivity is not critical the reaction time may be reduced to 18 h or even less. Testing serial dilutions of human convalescent antiserum against hepatitis A virus by ISEM showed the well known prozone effect with low binding of virus at low dilutions of serum, maximum binding at dilutions 1: 800 to 1 : 3,200 and decreasing virus trapping at increasing serum dilutions. A comparison of the four methods applied to detect hepatitis A virus in sucrose gradient

fractions

showed that ISEM and RIA were essentially

equal in sensitivity

and

superior to both direct EM and conventional IEM. The amount of virus particles demonstrated by ISEM and virus antigen determined by RIA in the gradient fractions are on the whole in good accordance. The low values measured for hepatitis A antigen by RIA in fractions 5 and 6 are difficult to explain, Possibly the particles in these fractions reacted poorly with specific IgM. Conventional IEM is less sensitive than ISEM and RIA and less reliable. Fractions which contain a high number of virus particles as shown by ISEM, as for example fraction

9, are negative

demonstrated

by ISEM,

by IEM, while fraction is positive

3 in which only a few virus particles

by IEM. This makes it difficult

fractions. Direct EM has a very low sensitivity.

Only the top fraction,

and RIA, is positive by this method. Immunosorbent electron microscopy

is a simple method,

to locate the top

as determined neither

are

by ISEM

dialysis nor cen-

trifugation steps are necessary. Only small samples are needed, about 10 ul, which is essential as usually small amounts of material are available when working with gradient fractions. The test may be performed within a reasonable period of time. We find adsorption overnight at 4°C convenient but previous work has shown that 3-4 h incubation at 33°C resulted in the same number of particles trapped on the grid (Kjeldsberg and Mortensson-Egnund, 1982). If an electron microscope is available ISEM thus seems to be a suitable method for demonstration of virus particles in sucrose

solutions.

167

ACKNOWLEDGEMENTS

We thank supplying

Professor

M. DegrC for reviewing

the hepatitis

A virus-infected

the manuscript

and Dr. E. Tj&ta

for

cell cultures.

REFERENCES

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D. and

Maramorosch

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E., 1977, Acta Pathol.

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D.E., R.F. Bozarth

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