Detection of rota virus immune complexes: Relationship between rotavirus antibodies and rota virus antigens in faeces

Journal of Virologikal Methods, ElsevierlNorth-Holland

3 (1981)

Biomedical

DETECTION OF ROTAVIRUS ROTAVIRUS

271-

217

282

Press

IMMUNE COMPLEXES: RELATIONSHIP

ANTIBODIES AND ROTAVIRUS

BETWEEN

ANTIGENS IN FAECES

G. CORTHIER C.N.R.Z., (Accepted

Laboratoire d’Ecologie Microbienne, 17 August

An ELISA

France

1981)

was developed

The relationship

78350 Jouy-en-Josas,

between

for

rotavirus

the identification antigens,

of rotavirus

rotavirus

immune

immune complexes

complexes and rotavirus

in pig faeces. anbibodies

of IgA class was examined.

rotavirus

immune

complexes

INTRODUCTION

The diagnosis of rotavirus infections is mainly based on virus or viral antigen detection in faeces (Bohl, 1979; Flewett and Woode, 1978; Narang and Codd, 1980). Recently, rotaviral antibodies have been found in human and pig faeces (Watanabe and Holmes, 1977; Watanabe et al., 1978; Corthier and Franz, 1980). Such antibodies may mask rotavirus in immunological tests used for its identification. Thus the detection of rotavirus immune complexes (IC) should be carried out for diagnosis. Recently, an ELISA was developed for the detection of IC (IC-ELISA) formed with tetanus toxoid, hepatitis B surface antigen (Pernice and Sedlacek, 1979) and parasites (Petit et al., 1977). In the present

study,

a similar IC-ELISA

is described,

based on the assumption

that

rotavirus antigenic sites are not completely engaged in antibody reaction. Thus, rotavirus IC may be adsorbed by means of antibodies against rotavirus to a solid phase by their free antigenic determinants and then quantified in situ by their content of pig immunoglobulins

(Ig) (Fig. 1). The relationship

between rotavirus, rotavirus IC and rotaviral anti-

bodies was examined in pig faeces. MATERIALS

AND METHODS

Biological samples Faeces from healthy pigs were collected during the winter of 1979-1980 in a conventional pig farm where no clinical signs of diarrhoea were observed in pigs of any age. 0166-0934/81/0000-0000/$02.75

@Elsevier/North-Holland

Biomedical

Press

278

EN

T Pig L lg

Fig. 1. Rotavirus-specific anti-rotavirus;

R, rotavirus;

detection

of soluble

E, alkaline

immune

phosphatase;

complexes

(IC): principle

Z Pig L Ig, antibody

against

of the method

(ZR,

pig immunoglobulin

light chain).

Nevertheless, 90% of these animals had serum antibodies against rotavirus (Corthier et al., 1979). Faecal samples from 23 pigs with diarrhoea were collected from 23 different farms in Brittany. All faecal samples were diluted l/l0 in phosphate buffered saline containing 0.05% Tween 20 and 0.02% sodium azide (PTSA). Faeces were clarified at 3500 X g for 30 mm. Pig rotavirus was extracted from faeces of pigs with diarrhoea and purified as described previously (Cohen, 1977). Hyperimmune sera specific for pig rotavirus were obtained from specific pathogen-free pigs (Corthier et al., 1980). Rotavirus rabbit IgCs were prepared by ion exchange chromatography (Harboe and Ingild, 1973) from sera of rabbits hyperimmunized with the purified pig rotavirus (OSU strain) (Bohl, 1979). Specific rabbit Ig or pig Ig light-chain antibodies or antisera to pig IgA heavy chains were obtained as previously described (Corthier and Franz, 1980; Franz and Corthier, 1980). IgA purified by ion exchange chromatography and gel filtration was used for the immunization of rabbits. Anti-light chain activity of hyperimmune sera was adsorbed on a Sepharose IgG polymer and eluted at pH 2.8. The specificities of the antisera were tested by immunoelectrophoresis and ELISA. Coupling of antibodies with alkaline phosphatase was carried out by the method of Avrameas and Ternynck (1969). The conjugates were used at a concentration

of 5 pg/ml.

At working

dilutions,

the antisera and conjugates

were devoid of any rotavirus activity. EL ISA

The methods

for detection

of rotaviral

antigens

and rotaviral

IgA antibodies

were

described previously (Scherrer and Bernard, 1977; Seigneurin et al., 1979; Corthier and Franz, 1980). The last procedure may be summarized as follows: microplates were coated with purified single-shelled rotavirus and incubated with the unknown samples. Antirotavirus IgA were shown with rabbit anti-IgA heavy chain and pig anti-rabbit immunoglobulin coupled to alkaline phosphatase.

279

The detection

of rotavirus

IC was performed

as follows:

microplate

wells (Linbro

polystyrene plates) were treated with 1% glutaraldehyde (1 h at 4°C) washed thoroughly with water, and coated with 100 ~1 of rabbit anti-rotavirus IgG (5 pg/ml in 0.05 M carbonate buffer, pH 9.6). The negative controls were tested by using purified normal rabbit IgG (devoid of anti-rotavirus activity when diluted at 5 pg/ml) for coating of the microplate. The plates were incubated for 3 h at 37’C and then rinsed three times with PTSA. Samples to be tested were diluted serially three-fold in PTSA, and 100 ~1 of each dilution added to each well. After overnight incubation at room temperature, the plates were washed three times and 100 ~1 of anti-pig light-chain conjugate was added. This conjugate had no anti-rotavirus activity at the working dilution. A further incubation period of 3 h at 37°C was followed by a three times wash, and a specifically absorbed enzyme was shown as reported before (Corthier and Franz, 1980). The results were expressed as an end-point titration, i.e. the highest sample dilution which yielded an optical density of 0.2 (background being 0.1). RESULTS

AND DISCUSSION

To determine the reliability of the IC detection test, the test was used in parallel with the direct ELISA (for the detection of free antigens) using artificial mixtures of a faecal rotavirus suspension and serial dilutions of an anti-pig rotavirus serum (Fig. 2). The rotavirus-serum mixtures were incubated for 1 h at 37°C and overnight at 4°C then examined by ELISA. At high concentrations of antiserum neither immune complexes (IC) nor rotavirus could be detected, probably as a result of the complete saturation of rotavirus antigens by specific antibodies. In a range of intermediate serum dilutions both immune complexes and rotavirus were detectable. At high serum dilutions little or no immune complexes were found and correspondingly the rotavirus particles became detectable. Similar results were obtained when purified pig or bovine rotavirus (single-shelled particles 0.3 pg/ml or 1.7 pg/ml) were used in the test (data not shown).

Fig. 2. Detection

of rotavirus

with serial dilutions of antiserum

concentration;

mean. In parentheses,

antibody

of an anti-pig rotavirus

-,

complexes

rotavirus detection

without

serum.

in artificial ---, Detection

of rotavirus

antiserum.

antigens

mixtures

of a pig rotavirus

of immune by the direct

complexes EL&A.

supension as a function

I, S.E. of the

280

The narrow dilution range where the IC test appeared significantly positive, while the direct ELISA appeared negative, was regularly observed. No visible precipitates were obtained after low centrifugation (3000 X g for 15 min) of any rotavirus-serum mixtures. The presence of rotavirus IC, rotavirus antigens and rotavirus IgA antibodies (aRIgA) were examined

in pig faeces collected

on 62 animals (23 pigs with diarrhoea

and 39

healthy pigs). The results were first interpreted qualitatively. A relationship was observed between rotavirus IC VS. rotavirus antigens (P < 0.001 x2 test, Yates correction; Table IA) and between rotavirus IC vs. aRIgA (P < 0.001; Table 1B). It should be pointed out that six faeces negative in the direct ELISA were found positive by IC-ELISA. The results could be interpreted quantitatively, when positive faeces titres are considered. A correlation exists between

rotavirus

IC and rotavirus

antigen

titre (P < 0.05; r =

-0.5). The titres

are inversely proportional (Fig. 3). Similarly, a correlation was observed between rotavirus IC and aRIgA (P < 0.01; r = 0.75) (Fig. 4). ELISA as reported here for rotavirus IC does not permit detection of saturated IC. For such complexes classical methods must be employed (reviewed by Lesavre and Mannick, 1980). Nevertheless, in the in vitro preparation of IC, rotavirus IC could be detected in an extensive serum dilution range, suggesting that unsaturated rotavirus IC occur rather frequently. In conclusion, the IC-ELISA for the detection of rotavirus IC is valuable for detecting the presence of rotaviruses in conditions where the direct assay yields doubtful results. Rotavirus antibodies of the IgA class only were studied, since it had been shown that this class of antibodies was predominant in pig faeces (Corthier and Franz, 1980). Detection of such antibodies may also be used for the characterization of rotaviral infections. However, the duration of rotavirus IgA production after a rotavirus infection is unknown. Without this information, the onset of rotavirus infection could not be estimated. The fact that a good correlation exists between rotavirus IgA and rotavirus IC suggests that formation of rotavirus IC occurred simultaneously with production of IgA antibody. Work is in progress to determine experimental rotavirus infection.

TABLE

the kinetics

of rotavirus

IgA and IC formation

after

1

Relationship

between

vs. rotavirus

IgA antibodies

rotavirus

IC vs. rotavirus

(aRIgA)

(B) (x’

antigens

(A) (x’

= 11.4; P < 0.001)

= 34.2; P < O.OOl).Titres

higher

than

and rotavirus

IC

10 are considered

positive B

A Rotavirus + Rotavirus

+

IC

Rotavirus +

9

6

6

41

aRIgA

t

IC

13

3

2

44

281

. . .

c

I 2

. 4

3

log Rotavirus titre

Fig. 3. Correlation

between

the logarithm

of anti-rotavirus

IC titre vs. logarithm

of rotavirus

antigen

of rotavirus

IC titre

titre (P < 0.05; r = -0.5).

/

a I

I

2

3

I .

log Rotavirus IC titre

Fig.

4. Correlation

(P < 0.01; r = 0.75).

between

the logarithm

of anti-rotavirus

titre

vs. logarithm

282

ACKNOWLEDGEMENT

I wish to thank Dr. Petit and Dr. Scherrer for helpful discussion

and Dr. Laporte for

critical reading of the manuscript. REFERENCES Avrameas,

S. and T. Ternynck,

1969, Immunochemistry

Bohl, E.H., 1979, J. Am. Vet. Med. Assoc. Cohen,

J., 1977, J. Gen. Virol. 36,395-402.

Corthier,

G. and J. Franz,

Corthier,

G., J.F. Vautherot

Corthier,

G., J. Cohen

Flewett, Franz,

6,53.

174,613.

1980, Infect.

Immun.

and P. Vannier,

and R. Scherrer,

T.H. and C.N. Woode, J. and G. Corhier,

31, 833.

1979, Ann. Rech. Vet. 10,65.

1980, Ann. Rech. Vet. 11,45.

1978, Arch. Virol. 57, 1.

1980, Clin. Exp. Immunol.

44,645.

1973, Stand.

2,161.

Harboe,

N. and A. Ingild,

Lesavre,

P.H and M. Mannick,

J. Immunol.

1980, Workshop

held at the 4th International

Congress

of Immunology,

Paris. McNulty,

M.S., 1978, J. Gen. Virol. 40, 1.

Narang,

H.K. and A.A. Codd,

Pernice,

W. and H.H. Sedlacek,

Petit, A., S. Bernard, Scherrer, Seigneurin,

G. Luffau

R. and S. Bernard, J.M.,

Biomedicine

1980, Lancet,

1192.

1979, J. Immunol. and P. Pery,

1977, Ann. Microbial.

R. Scherrer,

Methods

28, 33.

1977, Ann. Inst. Pasteur

128 C, 937.

128,499.

M. Baccard-Longere,

0. Genoulaz,

C. Feynerol

31,99.

Watanabe,

H. and I.H. Holmes,

Watanabe,

H., I.D. Gust and I.H. Holmes,

1977, J. Clin. Microbial.

6, 319.

1978, J. Clin. Microbial.

7,405.

and J.P. Gout,

1979,