Immunoperoxidase assay for the detection of specific IgG antibodies to Hantaan virus

Journal

of Virological

Methods,

10 (1985)

53

53-58

Elsevier JVM 00358

IMMUNOPEROXIDASE ANTIBODIES

ASSAY FOR THE DETECTION

TO HANTAAN

and G. BEELAERT

Department

Institute

(Accepted

5 September

A technique, antibody

immunoperoxidase

employs

fluorescent

antibody

Hantaan

antibodies

IgG test

antibody

Nationalesrraat

antibody

155. B-2000

(IPA), was developed

glass slides with air-dried

Vero-E6 cells. Antibody

indirect

fluorescent

Medicine,

virus. The same protein A-peroxidaseconjugate

sera. The IPA technique Hantaan-infected

of Tropical

Antw,erpen.

Belgium

1984)

using indirect

to Hantaan

IgG

VIRUS

G. VAN DER GROEN of Virology,

OF SPECIFIC

titers detected

for the detection

of IgG

was used with mouse, rat and human

gamma-ray-inactivated

and acetone-fixed

by IPA were comparable

to those detected by the

technique.

protein

indirect

A-peroxidase

Hantaan-infected

Vero-E6

immunoperoxidase

indirect

immuno-

cells

INTRODUCTION

Hemorrhagic

fever with renal syndrome

disease in humans with a variety forming a new genus (Hantavirus) the prototype

(Schmaljohn

(HFRS)

is a collective

of clinical symptoms in the Bunyaviridae

and Dalrymple

name adopted

for a

caused by a group of viruses family, Hantaan virus being

1983; Schmaljohn

et al., 1984). The symp-

toms in many parts of the world where HFRS is known to occur are different; therefore, specific serologic testing for diagnosis of HFRS is recommended (Lgdevirta, 1982). The use of Hantaan-infected Vero-E6 cells as the antigen source in the indirect immunofluorescent antibody test (IFA) has proved to be a powerful tool in the diagnosis of HFRS because of the strong epidemiological correlation between subclinical, mild and severe forms of human HFRS and subsequent anti-Hantaan antibodies (Schmaljohn and Dalrymple, 1983).

development

of

Hantaan antibodies have been detected in a large variety of animal species (Desmyter et al., 1983; Tkachenko et al., 1983), and sero-epidemiologic screening required the availability of many antispecies conjugates. In the present study a simple immunoperoxidase assay (IPA) was developed using the same conjugate for the detection of Hantaan IgG antibodies in human and animal

0166-0934/85/$03.30

% 1985 Elsevier

Science Publishers

B.V. (Biomedical

Division)

54

sera of different

species. This was compared

with the immunofluorescence

antibody

assay (IFA). MATERIALS

Hantaan

AND METHODS

antigen slide preparation

Hantaan-infected

Vero-E6

cells were prepared

as described

by McCormick

et al.

(1982). Gamma-irradiated (3 X 1G6RAD) inactivated Vero-E6 cell suspension infected with the 22nd passage of Hantaan virus strain 76-118, A549 (1 lth passage) stored in growth medium containing 10% dimethylsulfoxide at -20°C was used for slide preparation (Van der Groen et al., 1983a). After thawing the cells were washed three times by centrifugation (580 Xg, 5 min) with phosphate-buffered saline (PBS), pH 7.2, and resuspended in freshly membrane filtered 5% fetal calf serum saline solution. The washed cell suspension containing 5 X lo5 infected cells/ml was mixed with 5 X lo5 uninfected cells/ml in a ratio to obtain 30-40% of the total number of cells being infected with Hantaan. The cells were mixed gently with a pipet and transferred in a plastic container (Titertek Cat No. 77824-01). With a multichannel micropipet (Titertek Cat No. 77824-01) five drops (0.02 ml/drop) at a time were applied on alcoholcleaned Teflon-coated multispot slides (10 spots/slide, type SM 11 Wellcome). After three or four slides, the cell suspension was thoroughly mixed, to guarantee an homogenous distribution of cells. Slides were dried overnight at room temperature, fixed in acetone for 10 min, placed in plastic slide boxes and stored at -20°C until use. The same antigen slides were used in parallel experiments for both the immunoperoxidase and immunofluorescence techniques. Lot numbers 421 5-4-81 and 451 3-5-81 were used in this work. Indirect

IFA

immunofluorescent

was performed

antibody assay (IFA)

as described

by Johnson

et al. (1981).

Fluorescein-labeled

sheep anti-human IgG (Wellcome) Lot No. K 6753, sheep anti-mouse IgG (Pasteur) Lot No. 1274641, and rabbit anti-rat IgG (Nordic) Lot No. lo-375 were diluted respectively concentration

1: 80,l drop/ml

and 1 : 80 in PBS, pH 7.2, containing

of 0.1%. Sera used for testing were obtained

from a mild form of HFRS

in Belgium

(Van der Groen

Evans blue at a final

from humans

convalescing

et al., 1983), Great Britain

(Lloyd et al., 1984) and France (Dournon et al., 1984). Mouse sera used were from wild Clethrionomys glareolus, captured in the northern part of Belgium (Van der Groen et al., 1983). Rat sera were obtained from infected laboratory rats (Desmyter et al., 1983). Indirect

immunoperoxidase

antibody assay (IPA)

Stored slides were thawed, dried by air and covered with test serum. After incubation at room temperature for 120 min in a moist atmosphere the slides were washed three times (3 min each) with PBS, and dried. The slides were incubated for 120 min

55

with protein dilutions

A-peroxidase

(Sigma Lot No. 62F3936)

used were 1 : 40, 1 : 80 and 1 : 20 for analysis

respectively.

After washing

diluted

in PBS. The conjugate

of human,

three times with PBS the enzymatic

using a freshly prepared

substrate

dine-tetrahydrochloride

(Fluka

solution.

mouse and rat sera, activity

was detected

This was made of 5 mg 3,3’diamino_benzi-

AG) dissolved

in 10 ml Tris-HCl

buffer, pH 7.6, and

0.1 ml H,O, 1% solution in distilled water. After shaking vigorously the solution was filtered (Whatman No. 1) and a colourless clear substrate solution was obtained, which was added on the antigen slides for 10 min at room temperature. After washing three times (3 min each time) the slides were mounted with glycerol phosphate, pH 7.4, and read with an oil immersion light microscope (X 1,000). Dark brown characteristic granular cytoplasmic staining of Hantaan antigen inclusions was obtained. A serum was considered positive when characteristic inclusions occurred in at least 10 infected cells per spot. The lowest dilution used for screening was 1: 16. Each positive serum was retested at least once on antigen-containing slides as well as on slides containing non-infected cells, to exclude any aspecific reaction. The IPA test was also performed using goat anti-human

IgG-peroxidase

(Nordic

Lot No. 371180), goat anti-mouse IgG peroxidase (Nordic Lot No. 26882) and rabbit anti-rat IgG peroxidase (Nordic Lot No. 7381) at 1 : 320, I : 40 and 1 : 80dilution,being the optimal conjugate dilutions. When antispecies peroxidase-labeled conjugates were used, the slides were incubated 30 min at room temperature with 1 : 10 diluted normal goat serum (when conjugate prepared in goats was used) or normal rabbit serum (when conjugate prepared in rabbits was used). After washing the slides once with PBS and drying, the conjugates were added and processed as described before. RESULTS

Fig. Vero-E6

la shows

the typical

cells by peroxidase

the peroxidase

reaction Hantaan

cytoplasmic with a positive

because infected

a ratio 1: 3 to 1 : 4. Cytoplasmic cells or when

granular reaction staining

antibody-negative

inclusions

of Hantaan-infected

serum. Not all the cells showed

cells have been mixed with uninfected was completely

absent on uninfected

sera were investigated

(Fig.

cells in Vero-E6

lb). In the

absence of any trace of serum no aspecific binding of protein A-peroxidase occurred on Hantaan-infected and uninfected Vero-E6 cells, in contrast to the peroxidase-labeled antispecies conjugates. The latter also showed a more pronounced aspecific binding on uninfected cells when sera were tested. With final concentrations 0.03%, 0.06% and 0.09% of H,O, in the substrate solution a 2-4-fold decrease in titer of a positive serum was obtained. A decrease (5 min) or increase (20 min) of substrate incubation times did not significantly change the titer of a positive serum. The use of benzidine (Cevenini et al., 1983) instead of 3, 3’diamino-benzidine-tetrahydrochloride failed to give reproducible results. The optimal protein A-peroxidase conjugate dilution was determined each time a new lot of conjugate was used as well as each time sera of another species were

*

.

Fig. 1. A typical infected

dark granular

and (b) uninfected

4

5

6

7

R

cytoplasmlc

Vera-Eh

9

stammg

of Hantaan

cells by the protein

10 11 12

4

5

6

7

8

IFA titer

Fig. 2. Comparison

of antibody

oxidase

and

conjugate

conjugates.

titers detected

by indirect

9

10 11 12

serum on (a) Hantaan-

reaction

4

5

6

(X324).

7

8

9

10 11 12

(log,)

by indirect

immunofluorescence

antibody-posltlve

A-peroxidase

immunoperoxidase assay

(IFA)

using

(IPA) usmg protein antispecies

A-per-

FITC-labeled

57

analysed.

The optimal

1 : 20, respectively.

1 : 80 and two-step

conjugate

dilution

dilutions

for human,

The optimal

below the highest dilution

mouse and rat sera were 1 : 40,

conjugate of conjugate

had still the highest titer and no aspecific binding

dilution

was defined

for which a positive

of conjugate

as one serum

in the absence of serum

on infected and uninfected cells occurred. Sera of 54 men, 23 laboratory rats and 37 wild mice (Clethrionomys glareolus) were examined by both IPA and IFA. Fig. 2 demonstrates the comparison of IPA and IFA titers. Incubation times of serum and conjugate shorter than 120 min resulted in a significant

lowering

of titer in the IPA test.

DISCUSSION

With better diagnostic methods, Hantaan has been found to be a widespread pathogen, involved in many human infections. The presence of Hantaan antibodies in many different animal species (Tkachenko et al., 1983) required the availability of many antispecies conjugates. In the present study we developed an indirect peroxidase antibody technique to Hantaan. The IPA described was as sensitive as the IFA technique, for each of the different groups of sera tested. A good correlation (r = 0.969, 0.889, 0.993) was obtained for human, mouse and rat sera, respectively. The titer was reproducible within a 2-fold range. The IPA technique using protein A-peroxidase as conjugate could detect the low level of IgG antibody to Hantaan in the normal population as well as the high titers of clinically well documented HFRS cases and of infected laboratory rats. The results also showed that background staining was completely absent when protein A-peroxidase was used. Antispecies conjugates presented serious problems of unwanted staining and required adsorption with noninfected Vero-E6 cells in order to reduce or abolish this. The protein A-peroxidase conjugate obviates the laborious and time-consuming immunization procedure each species the optimal

that is required to obtain antispecies immunoglobulins. For protein A-peroxidase conjugate dilution must be determin-

ed. Protein A isolated from the cell walls of Staphylococcus aureus is known to bind specifically to the Fc region of immunoglobulin G (with the exception of subclass 3) from several species with different affinities (Richman et al., 1982). Therefore the present

method

can possibly

be used to screen the sera of a much wider variety

animal species using one and the same conjugate. Each time optimal dilution and serum/conjugate incubation times should be determined. The method is suitable for large-scale screening of Hantaan antibodies.

of

conjugate The Han-

taan-infected Vero-E6 cell suspensions can be stored for months at -20°C with preservation of their antigenicity and the peroxidase activity on the slides was easily read by low-power light microscopy. There is no need for an expensive UV light microscope. The method is simple and sensitive and has the potential for a widespread application. However, the technique will be of no use in the diagnosis of acute infection when the antibody may be expected to be predominantly IgM, because protein A-enzyme conjugates bind preferentially IgG.

58

ACKNOWLEDGEMENTS

by a grant from the Fonds Geneeskundig

This work was supported onderzoek,

contract

M. Jamaer,

J. van Hooren,

Lloyd, P. Sureau, the collection

Wetenschappelijk

No. 3000178. We would like to thank C. van Ypersele de Strihou, E. Callewaert,

J. Desmyter,

of human

E. de Potter,

F. Brasseur,

and animal

C. Deckers,

E. Lammerant,

L. Muylle, G.

H. Bazin and R. Verhagen

for

sera.

REFERENCES

Cevenini,

R., Rumpianesi,

Desmyter.

J., Johnson,

Dournon,

E., Moriniere,

Johnson,

K.M., Elliott,

Llhdevirta, Lloyd,

G., Bowen,

Schmaljohn, Tkachenko,

C., Leduc,

B. Matheron

E.T.W.,

Cleveland,

J. Infect. Jones,

D.L..

Dis. Suppl.

N. and Pendry.

1981, J. Clin. Microbial. A.. 1984, Lancet

I, 1175.

K.M.,

M.N. and Johnson, 1983, Virology J.M.,

14, 527.

36, 93.

P.H., Oxman,

S. and Dalrymple,

2. 1445.

1, 676.

1982, Lancet

J.M.,

36, 353.

F., 1983, Lancet

E.L., Sasso, D.R. and Kiley, M.P.,

C.S. and Dalrymple, C.S., Hasty,

I., 1983, J. Clin. Pathol.

J.W. and Brasseur,

S. et al., 1984, Lancet

L.H. and Heymann,

J.B., Palmer,

D.D.,

Schmaljohn, March

M. and Sarov,

J., 1982, Stand.

McCormick, Richman

F., Donati K.M., Deckers,

I. 765.

1982, J. Immunol.

128. 2300.

131, 482.

1984, Arthropod-borne

virus information

exchange,

12, 158. E.A., Ivanov,

A.P., Donets

van der Green.

G., Kurata

van der Green,

G.. Piot, P., Desmyter

van der Green,

G., Tkachenko,

et al.. M.A.,

1983, Ann. Sot. Beige Med. Trop.

T. and Mets, C. 1983a, Lancet

et al.. J., 1983b, Lancet

E.A., Ivanov.

63, 267.

1, 654. 2, 1493.

A.P. and Verhagen,

R.. 1983c, Lancet

2, 1 IO.