Journal of Virofo@al Methods, 11 (1985) 199-206

Elsevier

JVM 00414

199

DETECTION OF ROTAVIRUS-SPECIFIC IgG ANTIBODIES BY

IMMUNOPEROXIDASE ASSAY AND ENZYME-LINKED IMMUNOSORBENT

ASSAY

BAT-SHEVA ZENTNER’, MIRIAM MARGALITH’, AARON GALIL2, BENJAMIN HALEVY’

and ISRAEL SAROV’,’

‘Virolqy Unir and 2Department of Pediatrics, University Cenrer for Health Sciences and Soroka Medical

Center, Ben Gurion University of the Negev, Beer Sheva, Israel

{Accepted 22 February 1985)

An indirect immunoperoxidase assay (IPA) has been developed for determination of IgG antibodies to

rotavirus. The technique employed as antigen, SA-I 1 infected MA 104 cells, which were air-dried on glass

slides and acetone-fixed. In parallel, rota-specific IgG antibodies were determined by enzyme-linked

immunosorbent assay (ELISA). Specific IgG antibodies to rotavirus were determined in sera of healthy

children andinseraofpatientssufferingfromgastrornteritis. Agoodcorrelation (r=0.92)and(r=0.98)for

healthy children and patients, respectively, was found between IPA and ELISA techniques.

The IPA technique is rapid and simple and positive results, because of the intensive staining, are easily

read by low-power light microscope. The potential application of IPA and ELISA methods in serodiagnosis

of rotavirus infections is discussed.

rotavirus IgG immunoperoxidase enzyme-linked immunosorbent assay

INTRODUCTION

Various serological methods have been developed for the detection of antibodies to

rotavirus. These methods include a neutralization test (Thouless et al., 1977),

immunoeiectron microscopy (IEM) Kapikian et al., 1974), counter immunoeie~tro-

phoresis (CIEOP) (Cook et al., 1978), indirect immunofluorescen~e (IF) (Blacklow,

1976), complement fixation (CF)(Gust et al., 1977), hemagglutination inhibition assay

(HAI) (Martin et al., 1979) and enzyme-linked immunosorbent assay (ELISA)

(Yolken et al., 1978). These methods, although sensitive, have the disadvantage of

requiring equipment not found in all routine laboratories. Recently, we have devel-

*Address for correspondence: Professor I. Sarov, Virology Unit, Faculty of Health Sciences, Ben Gurion

University of the Negev. P.O. Box 653, Beer Sheva 84 10.5, Israel.

0166-0934/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

200

oped a simple immunoperoxidase assay (IPA), which utilizes virus infected target cells

air-dried and acetone-fixed, on glass slides, which can be used whenever rapid

laboratory diagnosis is desired. This technique is simple, rapid, specific, sensitive and

requires only a light microscope and equipment found in any laboratory. The IPA has

been adapted for the detection of IgG antibodies to measles virus, IgG and IgA

antibodies to herpes simplex virus, varicella zoster virus, cytomegalovirus, Chlamydia

trachomaris and human spermatozoa (Sarov and Haikin, 1983; Hadar and Sarov,

1984).

In the present study we adapted IPA for the detection of IgG specific rotavirus

antibodies. The results obtained by the IPA technique were compared with those

obtained by ELISA.

MATERIALS AND METHODS

Cells and media

MA 104 embryonic Rhesus monkey kidney cells (Microbiological Associates, Be-

thesda, MD) were grown in RPM1 1640 medium (Roswell Park Memorial Institute)

supplemented with 10% fetal calf serum (FCS) (Gibco, Grand Island, NY), 1%

glutamine, 100 U/ml penicillin, 100 ug/ml streptomycin and 10 U/ml mycostatin

(antibiotic antimycotic solution 2; Biolab, Jerusalem).

Rotavirus antigen preparation

Confluent, 3-day-old, MA 104 cultures were washed and infected with simian

rotavirus SA-11 (Malherbe and Strickland-Cholmley, 1967; Estes et al., 1979). To

enhance virus infectivity, virus was pretreated with 5 ug/ml trypsin for 1 h at 37°C

(Almeida et al., 1978). Virus stock at a 1 : 50 dilution in RPM1 was adsorbed to MA 104

monolayers for 1 h at 37°C. After incubation, the virus was removed and the cultures

were covered with RPM1 1640 supplemented with 2.5 ug/ml trypsin. When the cells

exhibited a mild cytopathic effect, 24 h later, the antigen was ready to be processed,

either to serve for the IPA or the ELISA technique.

Antigen for IPA: The cells were removed with a standard solution of 0.02% versene,

0.25% trypsin, and washed twice with 0.01 M phosphate buffered saline (PBS), pH 7.3.

Drops of infected cell suspension containing lo6 cells/ml were placed on glass slides,

dried at room temperature, fixed for 5 min in acetone, and stored at -70°C without loss

of activity for 6 mth. In parallel, control slides were prepared from uninfected sister

cultures.

Antigenfor ELISA: The infected monolayers were washed twice in PBS, and frozen at

-70°C with a minimal amount of PBS. Freezing and thawing was repeated three times

and was then followed by sonication in a Bransonic 12 sonifier for 1 min. The

suspension was centrifuged at 1,400 X g for 15 min at +4”C to remove cellular debris

and the supernatant was used as antigen. Protein concentration of the antigen prepara-

201

tion was determined by the method of Lowry et al. (1951). This antigen was stored at

-70°C.

Immunoperoxidase assay (IPA)

Stored slides were thawed and washed with PBS. Drops of serial two-fold dilutions

of test serum were placed on the antigen and incubated for 45 min at 37°C in a moist

atmosphere to avoid drying of the sera. The slides were washed with PBS and

incubated further for 15 min in PBS and then dried. Anti-human IgG peroxidase

conjugate (Miles Yeda, Kiryat Weizman, Rehovot, Israel) diluted 1 : 50 was added and

the slides were incubated for 45 min at 37°C. After incubation, the slides were washed

as described and dried.

The bound peroxidase enzymatic activity was determined using benzidine substrate

in a modification of the method of Graham and Karnowsky (1966). The benzidine

substrate was made fresh as follows: 4 mg benzidine was dissolved initially in 0.5 ml

acetone and 9.5 ml PBS (pH 7.3) and 10 ~1 of 33% hydrogen peroxide was then added.

The enzymatic reaction was allowed to proceed for 5 min after which the slides were

washed with PBS as described and dried. Stained cells were examined under ordinary

light microscope at X 25 and X 40 magnification. Infected cells which were incubated

with positive sera developed dark blue or brown stains in the cytoplasm. Approximate-

ly 30% of the cells were initially infected and ca. 1,000 cells were screened with each

serum dilution. In parallel, slides from uninfected control cells were also tested.

ELISA

The procedure is a modification of that described by Bidwell et al. (1977). Aliquots

of 5 ug protein of SA-11 antigen preparation or control antigen in 0.025 ml were

dropped into wells of the polystyrene microtiter plate (NUNC Microtest 96U-1182)

and allowed to dry overnight at room temperature. Before use the plates were washed

with PBS containing 0.025% Tween 20 (PBST) which was also used for rinsing the

plates after each step. Volumes of 0.025 ml of serial four-fold dilution of the sera in

PBS i- 0.5% bovine albumin (fraction V, Sigma, St. Louis, MO, U.S.A.) were dropped

into the appropriate wells and incubated at 37°C for 1 h. After the plates were rinsed

and dried, 0.025 ml anti-human IgG peroxidase conjugate (Dakopatts, Copenhagen)

at a 1 : 250 dilution was added and incubated at 37°C for 1 h; the plates were rinsed

again. The peroxidase activity was then followed by addition of the substrate solution,

0.1 ml distilled water containing 0.085% 5-amino salicylic acid with 0.005% H,O, (pH

6. l), to each well. The enzymatic reaction was stopped by adding 0.1 ml NaOH 1 N to

each well. Well contents were transferred to individual tubes, diluted with 1 mlofwater,

and absorbance at 450 nm was measured with a Gilford Microsample Spectrophoto-

meter (Mode1 Stasar II). Each serum was tested in parallel on control antigen prepared

from uninfected MA 104 monolayers, and each test included known positive and

negative sera. The serum titer was determined as the intersection between the serum

titration curve on viral antigen and the corresponding curve obtained with the control

antigen as described previously (Sarov et al., 1980).

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Human serum samples

Serum samples were obtained from two sources: (a) ninety-three randomly selected

samples from l- and 2-yr-old healthy children; and(b) samples obtained from patients

during an outbreak of rotavirus gastroenteritis in an agricultural community (kibbutz)

in the southern part of Israel (Galil et al., 1985). Rotavirus antigen was detected in all

first stool samples of these patients by ELISA applying Dakopatts enzyme immunoas-

say for the detection of rotavirus antigen (rotavirus ELISA) (Dako, Copenhagen,

Denmark) and by electron microscopy (Cevenini et al., 1984). All serum samples were

tested in parallel by the IPA and ELISA techniques. The paired serum samples from

each patient were always assayed together (Galil et al., 198.5).

RESULTS

Immunoperoxidase reactions

Figure 1 shows a positive IPA reaction with the typical intensive dark blue peroxi-

dase catalyzed staining in the cytoplasm of the rotavirus-infected MA 104 cells. These

cells have reacted with sera containing antibodies to rotavirus, and then were process-

ed by the IPA method. The bound antibodies were detected by peroxidase conjugated

anti-human IgG. Positive cells were very distinctive among the colorless uninfected

cells. Cytoplasmic staining was absent from uninfected MA 104cells tested in parallel.

Fig. I. lmmunoperox~dase staining of rotavirus-infected MA 104 cells, wth rotavirus IgG-positive serum.

203

The use of peroxidase-conjugated antibody to human IgG at a dilution of 1 : 50 gave

satisfactory results for the determination of titers of IgG antibodies to rotavirus.

Comparative analysis of ZgG titer by ZPA and ELZSA - healthy children

The sera of 93 infants and children were tested for the presence of IgG antibodies to

rotavirus by both IPA and ELBA. Figure 2 represents the comparison of titer of

antibodies obtained by the IPA and ELBA methods in serum samples from healthy

children. The calculated correlation coefficient between the two methods was found to

be r = 0.92. Sixty-two sera (66.6%) were found to be positive by IPA (2 2) and ELISA

(2 40) and thirty-one (33.3%) were negative.

Comparative analysis of ZgG titer by ZPA and ELZSA - patients

Two serum samples were collected from each of eight rotavirus patients. The first

samples were collected during the acute phase of illness and the second during

convalescence (Table 1). Paired sera from each patient were assayed together by IPA

and ELISA. The results indicate that a low titer of rotavirus IgG antibodies was

detected in the first serum sample tested by both methods, IPA and ELISA. The

second samples exhibited an increase in the titer of rotavirus IgG antibodies as

detected by both methods, IPA and ELISA. It is of interest to note that the serum

64-

32-

16-

I 40 160 I40 2550 0240 4cB6a

ELISA TITRE

Fig. 2. Comparison of rotavirus IgG antibody titers in serum samples from healthy children as determined

by IPA and ELISA.

204

TABLE 1

Comparison of IPA and ELISA antibodY titers in sera of patients with rotavirus infection.

Patient

no.

Age

(Yr)

Day 01

illness

Titer

IPA ELISA

I I Z/l2

II I ll/lZ

III 52

IV 34

V 5 9/12

VI 3 9/12

VII 52

VIII 25

2X

9

32

3

21

3

21

3

I6

1

23

2

24

3

I9

2 IO

8 640

12X 10,240

256 40,960

2 40

12X 10,240

I6 640

12X 10,240

2 40

I28 10,240

2 40

128 10.240

8 640

64 10.240

4 I60

12x 10,240

samples of patient no. 11 exhibited the highest titer in both methods. The serum

samples of this patient were taken later than all the others, 9 days and 32 days after the

onset of illness (Table 1). The calculated correlation coefficient for the results obtained

by the two methods in the tests of the patients’ sera was found to be r = 0.98.

Reproducibility All serum samples, the 93 from the healthy children and the 16 from the patients

suffering from diarrhea, were tested by the IPA and ELISA methods at least twice.

Many of the sera were examined several times. In all the instances the titer was

reproducible within a single dilution range.

DISCUSSION

Rotaviruses have been established as the major etiological agent of diarrhea in

infants and young children requiring admission to hospital for treatment of acute

gastroenteritis in many parts of the world. Studies on children admitted to hospitals

suggest that infection occurs most often between the ages of 6 mth and 3 yr (Bryden et

al., 1975; Esparza et al., 1977; Schoub et al., 1977a, b; Banatvala et al., 1978; Chrystie

et al., 1978; Flewett and Woode, 1978; Konno et al., 1978; Yolken, 1978; Brandt et

al., 1979; Soenarto et al., 1981; Lambert et al., 1983; Riepenhoff-Talty et al., 1983).

205

There are estimates that up to 50% of pediatric hospitalizations for acute gastro-

enteritis are due to rotavirus infection (WHO Scientific Working Group, 1980).

Rotavirus infections in healthy adults are generally asymptomatic or mild (Grimwood

et al., 1983; Smith et al., 1983).

Rapid and simple serological methods for the detection of rotavirus antibodies in

paired serum samples are a helpful tool for diagnosis of rotavirus infections since the

virus may be excreted in the patients’ feces for a relatively short time (Kapikian et al.,

1982). Such methods are also helpful for seroepidemiological surveys of the prevalence

of rotavirus infections.

In this report we have adapted the immunoperoxidase assay (IPA) for detection of

specific IgG antibodies to rotaviruses. The SA-11 monkey rotavirus was used as

antigen, since it is antigenically related to human rotaviruses and has been used for

estimation of immune responses to human rotavirus infections (Schoub et al., 1977;

Brade and Schmidt, 1979; Sheridan et al., 1981; Davidson et al., 1983; Smith et al.,

1983; Bishop et al., 1984). The SA-1 I virus relates to human rotaviruses belonging to

serotype 3 (Hoshino et al., 1984) and subgroup I (WHO Terminology, 1984) and

propagates easily in continuous cell lines as compared with most of the bovine

rotavirus strains. Although the titers of antibodies obtained by the IPA method are

lower than ELISA there was a good correlation as far as seropositivity is concerned

between the two techniques; sixty-two sera were found to be positive by IPA and

ELISA and thirty-one were negative. The correlation coefficient between the results

obtained by IPA and ELISA was r = 0.98 in rotavirus patients (Table 1) and r = 0.92 in

healthy children (Fig. 2). An increase in titer of IgG antibodies between the first and

second paired patients’ serum samples was demonstrated by both methods (geometric

mean titer 5.7 to 90.5 by IPA and 160 to 8,600 by ELISA).

In conclusion, both IPA and ELISA are reliable methods for the detection of

rotavirus specific IgG antibodies. The results of this study show that the IPA technique

is a quick, simple, reliable and sensitive method for the detection of rotavirus specific

antibodies. It requires only a light microscope for the detection of the intensive

staining of the positive cells. The IPA might be a very useful and convenient methodfor

serotyping viruses by serum neutralization. The advantage of the ELISA method over

IPA is that it can be automated, which, however, would require relatively expensive

equipment.

The future widespread use of IPA as a standard method in routine laboratories will

depend upon the stability of the antigen on slides, which can be transferred between

laboratories.

REFERENCES

Almeida, J.D., T. Hall, J.E. Banatvala, B.M. Totterdell and I.L. Chrystie, 1978, J. Gen. Viral. 40, 213.

Banatvala, J.E., I.L. Chrystie and B.M. Totterdell, 1978, J. Am. Vet. Med. Assoc. 173, 527.

Bidwell, D.E.. A. Bartlett and A. Voller, 1977. J. Infect. Dis. 135, 274.

206

Bishop, R.F., E. Cipriani, J.S. Lund, J.L. Barnes and C.S. Hoskmg, 1984, J. Clin. Microbial. 19, 447.

Blacklow, N.R., P. Echeverria and D.H. Smith, 1976, Infect, Immun. 13, 1563.

Brade, L. and W.A.K. Schmidt, 1979, Med. Microbial. Immunol. 167, 55.

Bran& C.F., H.W. Kim. R.H. Yolken. AZ. Kapikian, J.O. Arrobio, W.J. Rodriguez, R.G. Wyatt, R.M.

Chanock and R.H. Parrott, 1979, Am. J. Epidemiol. if0. 243.

Bryden, A.S., H. Davies, R.E. Hadley, T.H. Flewett, C.A. Morris and P. Oliver, 1975, Lancet ii, 241.

Cevenini, R., F. Rumpianesi, R. Marracchio, M. Donati, E. Palcieri and I. Sarov, 1984, .I. Infect. 8, 22.

Chrystie, I.L., B.M. Totterdell and J.E. Banatvala, 1978, Lancet i, 1176.

Cook, D.A.. A. Zbitnew. G. Dempster and J.W. Gerrard, 1978, J. Pediatr. 93, 967.

Davidson, G.P., R.J. Hogg and C.P. Kirubakaran, 1983. infect. Immun. 40. 447.

Esparza, J., B. Viera de Torres, A. Pinero, F.O. Carmona and R. Hazzali de Lija, 1977, Am. J. Trop. Med.

Hyg. 26, 148.

Estes, M.K., D.Y. Graham, C.P. Gerba and E.M. Smith, 1979, J. Viral. 31, 810.

Flewett, T.H. and G.N. Woode, 1978, Arch. Virol. 57, I.

Gal& A., R. Antverg, B. Zentner, M. Margalith, M.G. Friedman, B. Sarovand I. Sarov, 1985, Israel J. Med.

Sci., 21, 176.

Graham, R. and M.J. Karnowsky, 1966, J. Histochem. Cytochem. 14, 291.

Grimwood, K., G.D. Abbott, D.M. Fergusson, L.C. Jennings and J.M. Allan, 1983, Br. Med. J. 287.575.

Gust, I.D., R.S. Prinple. G.L. Barnes, G.P. Davidson and R.F. Bishop, 1977, J. Clin. Microbial. 5, 12.5.

Hadar, T. and I. Sarov, 1984, J. Med. Virol. 14, 201.

Hoshino, Y., R.G. Wyatt, H.B. Greenberg, J. Flores and AZ. Kapikian, 1984, J. Infect. Dis. 149, 694.

Kapikian, A.Z., H.B. Greenberg, R.G. Wyatt. A.R. Kalica, Hyun wha Kim,C.D. Brandt, W.J. Rodriguez,

R.H. Parrott and R.M. Chanock, 1982, in: Viral Infections in Humans Epidcmiolo~y and Controi, cd.

AS. Evans (Plenum Medical Book Company, New York) p. 283.

Kapikian, A.Z., H.W. Kim, R.G. Wyatt, W.J. Rodriguez, S. Ross. W.L. Cline, R.H. Parrott and R.M.

Chanock, 1974, Science 185, 1049.

Lambert, J.P., D. Mar&ens, P. Marbehant and G. Zissis, 1983, J. Med. Virol. 11, 31.

Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265.

Malherbe, H.H. and M. Strickland-Cholmiey, 1967, Arch. Gesamte Virusforsch. 22, 235.

Martin, M.L., G.W. Gary Jr. and E.L. Palmer, 1979, Arch. Virol. 62, 131.

Riepenhoff-Talty, M.. L.J. Saif, H.J. Barret, H. Suzuki and P.L. Ogra, 1983, J. Clin. Microbial. 17, 352.

Sarov, 1. and H. Haikin, 1983, J. Virol. Methods 6, 161.

Sarov, I., P. Andersen and K. Andersen. 1980, Acta Path. Microbial. Stand.. Sect. B 88, I.

Schoub, B.D., G. Lecatsas and O.W. Prozesky, 1977a. J. Med. Microbial. 10, I.

Schoub, B.D., A.S. Greeff, G. Lecatsas, O.W. Prozesky, I.T. Hay, J.G. Prinsloo and R.C. Ballard, 1977b.

J. Hyg. (Cambridge) 78. 377.

Sheridan, J.F., L. Aurelian, G. Barbour, M. Santoshan, R.B. Sack and R.W. Ryder, 1981, Infect. Immun.

31,419.

Smith, CC., L. Aurelian, M. Santoshan and R.B. Sack, 1983, Infect. Immun. 41, 829.

Soenarto, Y., T. Schoodo, R. Ridho, H. Alrasjad, J.E. Rhode, H.C. Buggs, G.L. Barnes and R.F. Bishop,

1981, 3. Clin. Microbial. 14. 123.

Thouless, M.E., AS. Bryden, T.H. Flewett, G.N. Woode, J.C. Bridger, D.R. Snodgrass and J.A. Herring,

1977, Archiv. Viral. 53. 287.

WHO Scientific Workrng Group, 1980. Bulletin WHO 58. 183.

WHO Terminology, 1984, Bulletin WHO 62. 501.

Yolken, R.H., R.G. Wyatt, H.W. Kim, A.Z. Kapikian and R.M. Chanock, 1978. Infect. lmmun. 19, 540.