Comparison of immunosorbent electron microscopy, enzyme immunoassay and counterimmunoelectrophoresis for detection of human rotavirus in stools

Journalof

VirologicalMethods,

EIsevier/North-Holland

COMPARISON

‘Groupe

99

ELECTRON MICROSCOPY,

AND COUNTERIMMUNOELECTROPHORESIS

ROTAVIRUS

G. OBERTI, R. GLOECKLER’, Facultk

99-107

OF IMMUNOSORBENT

IMMUNOASSAY OF H~AN

3 (1981)

Biomedical Press

de Recherches de MPdecine:

ENZYME

FOR DETECTION

IN STOOLS

J. BURCKARD’ and M.H.V. VAN REGENMORTEL’

SW In Pathogknie and ‘lwtitut

des Infections

Virales U 74, Laboratoire

de Biofogie MoEculaire

et Ceilulaire,

de Virologie,

15 rue Descartes,

67000

Stra~bourg, France

(Accepted 23 March 1981)

The detection

of human rotaviruses by routine electron microscopy examination

of stool speci-

mens has been compared with the sensitivity of detection obt~nable by three different immunoassays. These assays are: 1) immunosorbent electron microscopy (ISEM), which consists of the ~~ologi~d trapping of viruses on electron microscope grids coated with protein A and specific viral antiserum; 2) an enzyme-linked immunosorbent assay (ELISA), in which the primary antibody is rabbit antirotavirus immunoglobulin, the secondary antibody is chicken anti-rotavirus immunoglobulin extracted from egg yolk of immunized hens, and the indicator antibody is alkaline phosphatase-conjugated rabbit anti-chicken immunoglobu~n~ 3) counterimmun~electrophoresis (CIE). A total of 63 stool specimens from infants with gastroenteritis were examined. Of these, 23 and 24 specimens were found to contain rotavirus by eIectron microscopy and CIE, respectively. When scored by ELISA and ISEM, 37 and 39 were found to be positive, respectively. Confirmatory inhibition assays were necessary to eliminate some false positive reactions in ELISA. Detection of human rotaviruses in stools by ISEM is as sensitive as by ELISA, but in weakly positive specimens, ISEM offers the additional advantage of a direct visual demonstration of the presence of the aetiological agent. INTRODUCTION

Rotavirus children

infections

(McNulty,

in stool specimens

represent

one of the major causes of gastroenteritis

1978; Holmes, by electron

1979). The causative agent can be detected

microscopy

ex~nation

(Kap~ian

in young directly

et al., 1974), but

in many cases the virions can be visualized only after they have been concentrated by ultracentrifugation. Several serological techniques such as counterimmunoelectrophoresis (Middleton et al., 1976; Grauballe et al., 1977), enzyme-linked immunosorbent assay (Ellens and De Leeuw, 1977; Scherrer and Bernard, 1977; Zissis and Lambert, 1980), radio~unoa~ay (Kalica et al., 1977; Middleton et al., 1977; Sarkkinen et al., 1980), enzyme-linked fluorescence assay (Yolken and Stopa, 1979) and immunosorbent electron microscopy (Nicolai’eff et al., 1980) have also been used for detecting rotavirus in faecal material. Most of these immunological assays are more sensitive than routine electron microscopy, and also allow a more rapid detection of rotavirus in stool specimens. Ot66-0934/81/0000--0000!$02.5#

Q !+cv
, ~(~rt~~-~~l~~ll~.~~d Biomedical

Press

100

In the present study, we compared

the sensitivity

of detection

of rotavirus in human

stools by electron microscopy with the sensitivity achieved by three different immunoassays: immunosorbent electron microscopy (ISEM), an enzyme-linked immunosorbent assay (ELISA),

and counterimmunoelectrophoresis

1973; Roberts

and Harrison,

1979; Nicolaieff

(CIE). The ISEM technique et al., 1980) consists

(Derrick,

in the serological

trapping of viruses on electron microscope grids coated with specific ~tise~m against the virus. The particular ELISA method that was used utilizes chicken anti-rotavirus immunoglobulin extracted from egg yolk of laying hens immunized with rotavirus (Polson et al., 1980; Van Regenmortel and Burckard, 1980) and an alkaline phosphatase rabbit anti-chicken globulin conjugate. MATERIALS

AND METHODS

Specimens A total of 63 stool specimens were obtained from 63 infants admitted to the hospital in Strasbourg with symptoms of acute gastroente~tis. Four volumes of d~t~led water were added to one volume of faecal material and the mixture was homogenized and clarified by low-speed centrifugation (5000 g for 20 min). Purification

ofrotavirus

The procedure described by Rodger et al. (1975) was applied to a stool which had been found by electron microscopy to contain a large number of rotavirus particles (Nicolai’eff et al., 1980). The concentration of virus was expressed by the protein content determined by the method of Lowry et al. (195 1).

Rabbits

and laying hens were immunized

by a series of intramuscular

injections

of

rotavirus in adjuvant as described previously (Nicolaieff et al., 1980). Rabbit immunoglobulins were prepared by precipitation from antiserum with an equal volume of 4 M ~o~urn sulphate. Chicken ~munoglobu~s were obtained from the egg yolk of immunized hens as described previously (Polson et al., 1980). Routine electron microscopy The clarified stool extract was applied to carbon-coated electron microscope grids (300 mesh). After staining with 2% phosphotungstic acid (pH 7.0) the grids were screened for 15 min for the presence of rotavirus particles in a Philips EM 300 electron microscope at a magnification of 39,000 X . If a single particle was found, the specimen was scored as positive.

101

Immunosorbent electron microscopy Electron

microscope

pl drops of protein

grids, coated with a ffirn of formvar-carbon,

were floated on 50

A solution (26 pg/ml) for 4 min and were then placed successively on

three drops of 0.1 M sodium phosphate buffer (pH 7.0) for 1 min. Thereafter, the grids were floated for 10 min on 50 p.l drops of rabbit anti-rotavirus serum diluted 1 : 500. After an intermediate rinse on a drop of phosphate buffer, the antibody-coated grids were left overnight

on drops of stool extracts

diluted

with an equal volume of 0.2 M

phosphate buffer. After the serological trapping reaction, the grids were rinsed by placing them on a series of drops of distilled water. Virions were visualized by staining with 1% uranyl acetate in 45% ethanol for 2 min. Grids were screened for 5 min at an instrumental magnification of 8 100 X . Four areas were photographed with 70 mm film. Particle counts were made with a binocular

magnifier

on four 68 X 73 mm micrographs

(N’icolaieff et

al., 1980). Counterimmunoelectrophoresis The method used was similar to that described by Middleton et al. (1976). One percent agarose (Indubiose, Pharmindustrie, Clichy, France) in 0.05 M barbital buffer (pH 8.6) was applied to microscope slides. Stool extracts were placed in the cathode wells and specific rabbit antiserum in the anode wells. After electrophoresis at 10 V/cm for 2 h, the slides were washed in saline and stained with 1% tannic acid. Enzyme-linked immunosorbent assay An indirect ELISA method utilizing chicken and rabbit rotavirus antibodies was used (Scherrer and Bernard, 1977; Van Regenmortel and Burckard, 1980). Polystyrene microtitre plates (Cooke M 129 B, Dynatech) were used. Wells were coated at 37°C by 1 h incubation with 250 pl of rabbit immunoglobulins (10 pg/ml) diluted in 0.05 M sodium carbonate, saline, pH albumin in cubated at

pH 9.6. Plates were rinsed three times with 300 pl of phosphate-buffered 7.4, containing 0.05% Tween-20 (PBS-T) and once with 1% bovine serum PBS-T. Dilutions of purified rotavirus or of stool extracts in PBS-T were in37°C for 2 h in the coated wells. After rinsing, chicken immunoglobulins

(5 Erg/ml) in PBS-T were allowed to react at 37°C for 2 h with the trapped virus. After further rinsing, the wells were incubated for 2 h at 37°C with an anti-chicken globulin conjugate diluted 1 : 800. This conjugate was prepared by coupling IgG obtained from a rabbit immunized with chicken immunoglobulins with alkaline phosphatase (Boehringer, Mannheim), as described previously (VanRegenmortel and Burckard, 1980). After further rinsing, the bound enzyme conjugate was detected by adding 250 pl of the substrate p-nitrophenyl phosphate at 1 mg/ml in 0.1 M diethanolamine buffer, pH 9.8. After 1 h hydrolysis at 37”C, the tests were read with a photometer (Vernon, Paris) by measuring the absorbance at a wavelength of 405 m-n through the bottom of the microtitre plate.

102

Conjirrnato y ELISA In order to test the specificity absorbance kinen

and confirm

the validity of ELISA results in which the

values were lower than 0.7, a blocking

et al. (1980)

recently

showed

inhibition

the importance

test was performed.

of such confirmatory

Sark-

inhibition

tests to prove the specificity of low positive reactions. The blocking test was done as follows: 250 /_dvolumes of the specimens were added to three wells coated with rabbit anti-rotavirus globulins. After a 2 h incubation at 37”C, a 2.50 /.d volume of rabbit antirotavirus serum (1 : 200 dilution) was added to the first well and the same volume of normal rabbit serum (1 : 200) to the second well; the third one was filed with 250 fi PBS-T. After an incubation of 2 h at 37”C, the subsequent steps of the ELISA were performed as described above. The test was considered positive if a 50% or greater decrease in absorbance was obtained with the specimen incubated with rotavirus antiserum as compared with normal serum or dilution

buffer (Sarkkinen

et al., 1980).

RESULTS

The sensitivity of rotavirus detection by ELISA and ISEM was determined with a series of dilutions of purified virus, calibrated according to protein content. As shown in Fig. 1, as little as 2 ng/ml of purified rotavirus antigen could be detected by ELISA. In these tests, twice the buffer blank absorbance value was taken as the cut-off line. When the same dilutions of purified virus were examined by ISEM, the sensitivity of detection was essentially the same as by ELISA (Table 1). When 63 stool specimens were screened for the presence of rotavirus by four different

I

’ 1.25

’

2.5

1 5

ROTAVIRUS

Fig. 1. Sensitivity the means readings

’ 10

ANTIGEN

of detection

of readings

and corresponds

’ 20

from

’ 40 hg/ml)

of purified three

rotavirus

wells. The dotted

antigen

by ELISA.

line represents

to twice the value of the buffer

blank.

The absorbance

the cut-off

point

values

are

for significant

103

TABLE

1

Sensitivity

of rotavirus

detection

by immunosorbent

Rotavirus

antigen

40

electron

concentration

microscopy

(ng/ml)

20

10

5

2.5

1 0

Experiment

1

32a

25

11

3

2

Experiment

2

51

17

7

5

4

1

Experiment

3

47

23

10

7

3

0

130b

65

28

15

9

1

Total a

The values

for the three

separate

experiments

mm micrographs

taken at an instrumental

b

counts

Total

particle

represent

the total particle

counts

on three 68

x

73

of 8100 X

magnification

on nine micrographs.

techniques, the results presented in Table 2 were obtained. The sensitivity of detection by electron microscopy and CIE was similar and led to 36-38% of the samples being scored as positive. For these two techniques, the stool extracts were used undiluted and at a dilution of 1 : 2 respectively. By ELISA and ISEM, 59-6175 of the specimens were scored as positive when the stool extracts were diluted 1 : 20 and 1 : 2 respectively. The superiority of ELISA and ISEM was further confirmed by examining higher dilutions of the specimens by these two techniques TABLE

(Table 3). In the case of samples that contained

2

Comparison

of four methods

for detecting Rotavirus

No. of samples

rotavirus

in 63 stool specimensa

detection

by

Electron

CIE

ELISA

+

+

ISEM

microscopy 22

+

1

+

+ t

2

+ +

10 4

tb

2 22 Total:

63

100% a

Stool

CIE; 1 b

24

37

39

36%

38%

59%

61%

were examined

at the following

dilutions:

1

: 1 for electron

microscopy;

1

: 2 for

other

tech-

: 20 for ELISA, and 1 : 2 for ISEM.

Initially,

niques. c

specimens

23c

four samples

After confirmatory

The figures

represent

were scored ELISA

as positive

by ELISA

on these four samples

the total number

of samples

and as negative

by the three

(see Table 4) only two remained

scored

as positive

by each technique.

positive.

104

TABLE

3

Representative

results

weakly

stool specimens

Specimen

positive No.

of rotavirus

Method

of

detection

1

ELISA

0.875

ISEM

39

ELISA

0.65

ISEM a

Absorbance

b

Total

particle

1

451

ELISA

4

: 20

1

2.25

ISEM 3

of specimen

323b

ELISA

detection

Dilution

2.4a

ISEM 2

antigen

22

by ELISA

: 200 1.8

44 2.05 53

1

and ISEM with two highly

: 2000

1

: 20,000

1

and two

: 200,000

1.05

0.35

0.20

8

0

0

1.25

0.375

0.175

0

0

12

0.40

0.200

0.200

NTC

4

0

0

NT

0.35

0.200

0.200

NT

1

0

0

NT

at 405 nm. counts

on three

68 X 73 mm micrographs

taken at an instrumental

magnification

of

8100x. c

Not tested.

large numbers of particles, detection was still achieved at a specimen dilution of 1 : 2000, whereas by CIE and electron microscopy, the limit of detection was reached at a dilution of 1 : 20. In the case of weakly positive specimens, the limit of detection by ELBA and ISEM was reached at a dilution of 1 : 200. The absorbance values in the ELISA were then very close to the cut-off value for a positive result (Fig. l), whereas in ISEM, the low particle counts still allowed an unambiguous scoring of the specimens as positive. As shown in Table 2, four specimens were positive by ISEM and negative by ELISA, a finding which is in agreement with the slightly superior sensitivity of ISEM demonstrated in Tables 1 and 3. It was therefore somewhat surprising that the reverse situation was also found to exist, and that four specimens were initially scored as positive by ELISA and as negative by ISEM. In view of the possibility of non-specific binding in solid-phase immunoassays (Sarkkinen et al., 1980) these samples, together with all specimens with absorbance values below 0.7, were examined by confirmatory ELISA. Table 4 illustrates representative results obtained in four such confirmatory inhibition assays. Two of these tests were scored as negative, since they could not be specifically inhibited by rotavirus antiserum. DISCUSSION

Middleton et al. (1976) found CIE to be about 4-8 times less sensitive than electron microscopy, whereas Grauballe et al. (1977) reported that it was about 2--4 times more sensitive. Our own results agree with those of Tufvesson and Johnsson (1976) and Spence

10.5

TABLE 4 Confiimatory

ELBA inhibition test for establishing the specificity of rotavirus detection Absorbance when specimen incubated with dilution buffer

Absorbance when specimen incubated with rotavirus antiserum

Absorbance when specimen incubated with normal

2

0.85 0.75

0.325 0.275

0.75 0.85

3 4

0.55 0.50

0.175 0.225

0.200 0.200

Control antigen GO n&n)

2.15

0.75

1.85

Specimen No.

1

Interpretation of the test

SeNrn

+ +

+

et al. (1977) who reported that both techniques are about equally sensitive for detecting ro taviruses. Compared to electron microscopy and CIE, solid-phase immunoassays such as ELISA and radio~uno~say are generally considered to allow the detection of lower concentrations of viral antigens (Daugbarty and Ziegler, 1977; Middleton et al., 1977; Van Regenmortel, 198 1). Our own results (Table 2) confirm the findings of previous workers who showed that ELBA is a highly sensitive method for detecting rotaviruses in faecal extracts (Yolken et al., 1977; Ellens and De Leeuw, 1977; Scherrer and Bernard, 1977). The main d~advantage of solid-phase ~~unoassays lies in the possible occurrence of false positive reactions caused by non-specific binding (Sarkkinen et al., 1980). Since we did not observe any false positive reactions with specimens that gave ELISA absorbance values higher than 0.7 (all these specimens were positive by ISEM), we found it possible to eliminate non-specific reactions by performing confirmatory inhibition assays on all specimens with absorbance values below 0.7. The ISEM technique was equally sensitive as ELISA, but in weakly positive specimens, the direct visualization

of particles provided

a distinct

advantage

since no confirmatory

tests were necessary. When examing stool specimens by ISEM, we found that one microscopist could handle approximately 80 samples a day. Since microscope grids coated with protein

A and antiserum

can be stored for several weeks before use, they can be used

immediately as stool specimens become available. ~t~e~rn-coated grids can also be dispatched to centres that have no microscope facilities and returned for examination after completion of the trapping reaction. The trapping of virus particles on antiserumcoated grids occurs very rapidly and, with most samples, particles are visible after 60 min

incubation.

trapping Nicola’ieff

reaction

However, proceed

for maximum overnight.

sensitivity

As discussed

et al., 1980; Van Regenmortel,

of detection, previously

(Mime

198 l), the 1SEM procedure

we routinely

let the

and Luisoni, is simple

1977;

and rapid

106

and leads to fairly clean preparations, even when the stool extracts are allowed to react with the grids for 18 h. In weakly positive specimens, virus particles could be visualized within 5 min. The layer of serum proteins present on the grids prevents the adsorption of contaminants,

and as a result, small numbers

of virus particles are more easily visual-

ized than by routine electron microscopy. The primary coating of protein A on the grids considerably increases the efficiency of trapping by the adsorbed antibodies, since the IgG molecules are attached to the grid by their Fc regions and have their binding sites preferentially exposed (Shukla and Cough, 1979; Lesemann and Paul, 1980). The ISEM procedure has been used extensively in plant virology (Derrick, 1978; Milne and Lesemann, 1978; Roberts and Harrison, 1979; Lesemann et al., 1980; Nicolalieff and Van Regenmortel, 1980) and is also likely to find many applications in the diagnosis of human and animal virus infections. REFERENCI:S

Daugharty,

H. and

(Academic

D.W. Ziegler,

Derrick,

K.S., 1973, Phytopathology

Derrick,

K.S., 1978, Science PC.,

Holmes, Kaliq

J. Genner, R.H.

1977, J. Clin. Microbial.

A. Meyling

Purcell,

1977, J. Immunol. Kapikian,

A.Z.,

R.G.

1974, Science

and C. Kurstak

35,203.

M.M. Sereno,

R.G. Wyatt,

H.W. Kim, R.M. Chanock

and A.Z. Kapikian,

118, 1275. Wyatt,

W.J.

Rodriguez,

S. Ross,

W.L. Clin c, R.H.

Parott

and

R.M. Chanock,

185, 1049.

D.E. and H.L. Paul, 1980, Acta Hortic. D.E., R.F. Bozarth

and R. Koenig,

O.H., N.J. Rosebrough, MS.,

1977, J. Gen. Viral.

25, 1.

Lesemann, Lowry,

eds. L:. Kurstak

6,530.

and A. Hornsleth,

Lesemann, McNulty,

Diagnosis,

63, 538.

I.H., 1979, Prog. Med Viral. A.R.,

in: Comparative

199,538.

Ellens, D.J. and P.W. De Leeuw, Grauballe,

1977,

Press, New York) Vol. 2, p. 459.

110, 119.

1980, J. Gen. Viral. 48, 257.

A.L. Parr and R.J. Randall,

1951, J. Biol. Chem.

193, 265.

1978, J. Gen. Viral. 40, 1.

Middleton,

P.J., M. Pet&,

Middleton,

P.J.,

C.M. Hewitt,

M.D. Holdaway,

M.T. Szymanski

M. Petric,

and J.S. Tam,

M.T. Szymanski

1976, J. Clin. Pathol.

and J.S. Tam,

1977, Infect.

29, 191.

Immun.

16,

439. Mime, R.G. and E. Luisoni, (Academic

1977,

in: Methods

Milne, R.G. and D.E. Lesemann,

1978, Virology

Nicolaieff,

A. and M.H.V. Van Regenmortel,

Nicolafeff,

A., G. Obert

Polson, Rodger,

S.M., R.D. Schnagl

Scherrer,

R. and S. Bernard,

Shukla,

D.D. and K.H. Gough,

Spence,

L., M. Fauvel,

and PI<. Halonen, 1977,

1975, J. Viral.

1980, Immunol. 16, 1229.

1980, J. Viral. Methods

Ann. Microbial.

(Inst. Pasteur)

I, 33 I.

128 A, 499.

1979, J. Gen. Virol. 45,533.

R. Petro and S. Bloch,

B. and T. Johnsson,

1980, J. Clin. Microbial.

1979, Ann. Appl. Biol. 93, 289.

and I.H. Holmes,

H .K., H. Tuokko

Tufvesson,

and H. Koprowski

90,299.

and M.H.V. Van Regenmortel,

I.M. and B.D. Harrison,

Sarkkinen,

eds. K. Maramorosch

1980, Ann. Viral. (Inst. Pasteur)

and M.H.V. Van Regenmortel,

A., M.B. Von Wechmar

Roberts,

in Virology,

Press, New York) Vol. 6, p. 265.

1977, J. Clin. Microbial.

1976, Acta Pathol.

Microbial.

&and.

5, 248. B 84. 225.

13 1 L, 95. 12, 101. Commun.

9,475.

107

Van

Regenmortel, Wagner

(Plenum

Van Regenmortel,

M.H.V.,

1981,

M.H.V.

R.H. and P.J. Stopa,

Yolken,

R.H.,

1980, Virology

1979, J. Clin. Microblol.

H.W. Kim, T. Clem,

Virology,

and

R.R.

R.G. Wyatt,

106, 327.

10, 317.

A.R. Kalica,

2, 263.

Zissis, G. and J.P. Lambert,

eds. H. Fraenkel-Conrat

Vol 17, p. 183.

and J. Burckard,

Yolken,

Lancet

in: Comprehensive

Press, New York)

1980, J. Clin. Microbial.

11, 1.

A.Z. Kapikian

and R.M. Chanock,

1977,