JOIHAL OF I ICAL MEll#ODS

ELSEVIER Journal of Immunological Methods 2 12 ( 1998) 79-87

Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabies-neutralising antibody

F. Cliquet *, M. Aubert, L. Sag& CNEVA Nancy, Laboratoire d’e’tudes SW la rage et la pathologie des animaux sauoages, BP 9, 54220 Malzkcille, France

Received 29 October 1997; revised 20 November 1997: accepted 17 December 1997

Abstract

A microtest named the FAVN test (fluorescent antibody virus neutralisation test), which is an adaptation of the original rapid fluorescent focus inhibition test @FIT) has been developed and evaluated. One hundred percent specificity was estimated using 414 sera from dogs sampled in rabies-free areas or from non-vaccinated animals. The accuracy as determined by the agreement between observed and expected values using sera of known titres was satisfactory. Serum samples from unvaccinated and vaccinated dogs (using sera with titres near 0.5 IU/ml) were assayed for rabies antibody by the FAVN test, the RFFIT and the mouse neutralisation test (MNT): comparative results obtained on the same sera with the three tests showed good agreement. Furthermore, distinguishing negative sera from positive sera with low titres is much

easier with the FAVN test than with the RFFIT. 0 1998 Elsevier Science B.V. All rights reserved.

Keywords: Rabies neutralising antibody detection; FAVN test

1. Introduction

Since the first test developed in mice in 1935 (Webster and Dawson, 1935) for the detection of rabies virus neutralisation antibodies (MNT), several serological tests have been described including tests involving cell cultures (Smith et al., 1973; Zalan et al., 1979; Bussereau et al., 1982; Smith et al., 1996) and, more recently, an enzyme immunoassay

(Esterhuysen et al., 1995). The most commonly used technique is the rapid fluorescent focus inhibition test (RFFIT) described in 1973 by Smith, Yager and

* Corresponding author. Tel.: + 33-03-83-29-89-50: fax: + 33.

03-83-29-89-59; e-mail: fcliquet@cneva-nancy.fr

Baer, in which the determination of antibodies is indicated by a reduction in the number of fluorescent foci of virus-infected cells. This method was adapted for use in microplates in 1979 (Zalan et al., 1979) and involved, as did the original RFFIT developed in 1973, the counting of fluorescent foci. The purpose of this study was to investigate a microtest based on the principle of the RFFIT but using a fluorescence

read-out similar to the read-out usually employed for any other virological test. The new test, called the fluorescent antibody virus neutralisation test (FAVN

test), uses an 41 or nothing,, method of reading. This paper describes this technique and presents results obtained by the FAVN test in comparison with those obtained by the MNT and the RFFIT, which are both methods recommended by World

0022.1759/98/$19.00 0 1998 Elsevier Science B.V. All rights reserved.

PII SOO11- 1759(97)002 12-3

80 F. Cliquet et al. / Journal oj’Itnmunolopicn1 Methods 212 (1998) 79-87

Health Organisation (WHO) for the quantitation of rabies antibodies.

serum. Virus neutralisation titres were determined using the Spearman-KZrber method (Spearman, 1908; IGrber, 1931).

2. Materials and methods 2.3. FAVN test

Serum samples were taken from domestic dogs

currently vaccinated against rabies, laboratory dogs and unvaccinated dogs from both endemic and ra- bies-free areas. In order to assess the robustness of the FAVN test, sera of very poor quality were se- lected among fox sera sampled in the field. All sera

were heat-treated at 56°C for 30 min prior to testing and stored at - 20°C.

2.1. Reference sera

Each test included a positive and a negative con- trol. The standard used was the second WHO Stan- dard for Rabies Immunoglobulin (from the Statens Seruminstitut, Denmark) with an activity of 30 IU per ampoule (Lyng, 1994). The ampoule was recon- stituted with 1 ml of sterile distilled water and the

content transferred to a sterile tube. The ampoule was washed three times with 1 ml of sterile distilled

water, and the contents were added to the initial tube. A stock suspension was prepared to give a solution containing 6 IU/ml and then dispensed in aliquots of 1 ml and stored at -20°C. One aliquot

only was diluted to l/12 (titrating 0.5 IU/ml) and redivided into aliquots of 1 ml and stored at - 20°C. Each aliquot was sufficient for one week.

A pool of naive laboratory dog sera was included in each test as a negative control. This negative pool was divided into 0.5 ml aliquots lyophilised and stored at 4°C then reconstituted with 0.5 ml of distilled water before use.

2.2. Mouse h-us neutralisation test (MNT)

The MNT was performed in accordance with the method of Atanasiu (1966). To summarise, serial fivefold dilutions of sera, and of rabies standard serum containing 6 IU/ml, were tested against lo- 100 DL,,/0.03 ml of the challenge virus (CVS 27 strain). The virus-antibody mixtures were incubated for 1 h in a 37’C water bath, then injected intracere- brally (0.03 ml) into groups of 5 mice per dilution of

The FAVN test (Aubert et al., 1996) is an adapta- tion of the technique of Smith et al. (1973). It was

conducted on tissue culture 96-well microplates us- ing BHK21-13s (ATCC CCL-101 cells and the CVS 11 strain of rabies virus (ATCC VR 959). To prepare

the virus stock, suspended cells were infected with virus at a multiplicity of infection adjusted to 1 and incubated at 37°C for 40 h in 75-ml plastic flasks

containing growth medium, supplemented with 10%

heat inactivated fetal calf serum (EMEM-10% FCS). A large volume of virus was prepared and dispensed

in 0.5 ml aliquots stored at - 80°C. The titre of the virus was 105.9 TCID,,/ml. EMEM-10% FCS was used as a diluent for control and tested sera and for the challenge virus. Serial threefold dilutions of the positive and negative serum controls and of the

tested sera were made in the microplate wells in 0.1 ml volumes. Each serum was added to four adjacent wells and serially diluted four times using a multi-

channel pipette. A 0.05ml volume of a challenge virus suspension diluted in order to contain 50 to 200 TCID,,/well was added to each serum dilution well.

The microplates were then incubated for 60 min at 37°C in a humidified incubator with 5% CO,. Fol- lowing incubation, 0.05 ml of the cell suspension diluted in the growth medium to a concentration of 4 X 10” cells/ml was added to each well, and the plates were incubated for 48 h at 37°C in a humidi- fied incubator with 5% CO,. The controls used four

uninfected cell wells, back titration of rabies virus challenge dose, and the positive and negative refer- ence sera. After a 48-h incubation, the medium was

discarded into a virucidal solution, The plates were rinsed once in phosphate buffer saline (PBS) pH 7.2

(Dulbecco) and once in acetone (80% in distilled water). The plates were then fixed in 80% acetone (30 min, room temperature) and air-dried. The stain- ing was carried out by adding 0.05 ml of fluorescein isothiocyanate conjugated (FIT0 anti-rabies serum (Diagnostics Pasteur, France) to each well. After a 30-min incubation at 37”C, each plate was washed twice with PBS. Excess PBS was removed by briefly

F. Cliquet et al. /Journal of Immunological Methods 212 (1998) 79-87 81

inverting the microplates on absorbent paper. Results fluorescent cells in the virus control well, was added were assessed by using a microscope equipped for to each well. Following the neutralisation period for FITC fluorescence, with a X10 objective and a X10 60 min, 0.05 ml of BHK21- 13s cell suspension was ocular under X1.25 magnification. The total area of added to each well, and the microplates were incu- each well was examined. The well was considered bated for 24 h. The steps of fixation and staining negative if no fluorescent cells were observed. The were the same as those described in the FAVN test well was considered positive if one or more fluores- procedure. The microscopic reading of the fluores- cent cells were observed. The 50% endpoint of the cence was achieved by examination of the total antibody (D,o) content of test sera and virus titra- surface of each well, and then by recording the tions (TCID,,) were calculated according to the percentage of fluorescent cells. The titre of the serum Spearman-Karber method (Spearman, 1908; Karber, was the dilution at which a reduction of 50% of the 1931). The serum titres were expressed in IU per ml fluorescence was obtained. The number of ILJ per ml by comparison with the titre of the standard serum was determined by a comparison with the reference included in each test. serum.

The validity of each test was then assessed by recording results obtained from titrations of CVS

(TCID,,), naive serum (D,,) and the positive stan- dard (D,,) on control charts. Each test was consid- ered valid if the values found for all of these controls

were not statistically different from the respective means of the values obtained from all the previous tests.

The validity of each test was assessed using con-

trol charts as described above.

2.5. Statistical analysis

Statistical analyses were performed on the deci- mal logarithm (D,,) of the reciprocal dilutions with a 95% confidence limit, using linear regression and Student’s f-test.

2.4. RFFIT

The test was performed according to the tech- nique of Smith et al. (1973) modified by Zalan et al.

(1979) and Sureau et al. (1982). In brief, negative and positive controls and serum samples were di-

luted in the microplates in the same manner as described in the FAVN test, except that each serum

was tested in duplicate. A 0.05 ml dilution of virus stock (CVS 11 strain) suspension giving 80% of

Table 1

Use of FAVN test on 414 sera sampled from unvaccinated dogs

3. Results

3.1. Establishment of the threshold D5, of the FAVN test and RFFIT-control charts

A titre of 0.5 IU/ml is considered by the WHO (1992) as being the minimum protective antibody level, thus the reference standard serum diluted to

Status Origin Number of samples

Rabies-free areas

Endemic areas

Hawaii 42

Mauritius 39

New Caledonia 13

United Kingdom 30

France 111

France 10

France 144

North America 4

Tunisia 21

Dsa range (lowest-highest values)

0.24-1.19

<0.71-1.07

<0.71-<0.71

< 0.71-1.07

0.24- < 0.7 I 0.24-0.96

< 0.7 l-O.83

<0.71-<0.71

0.24- < 0.84

Mean (log, o )

0.59

< 0.72

<0.71

< 0.73

< 0.32

0.54

< 0.72

< 0.71

< 0.30

The D,,, are expressed as log,, values

82 F. Clique1 et al. / Journal of Immunological Methods 212 f 1998) 79-87

0.5 III/ml was used for each test. Each D,, value was reported on a control chart. The mean, calcu- lated from all the D,, data of the reference serum diluted to 0.5 RI/ml, and of the negative serum obtained with FAVN test were 1.52 (n. = 20, SD = 0.09) and 0.6 (n = 20, SD = 0.18>, respectively. The mean calculated from the D,, values when testing the reference serum diluted to 0.5 W/ml, and of the

negative serum by the RFFIT were 1.33 (n = 20, SD = 0.03) and 0.8 (n = 20, SD = 0.101, respec- tively.

A control chart was similarly used for the titration of the virus. Each RFFIT or FAVN test was consid- ered as valid only when the following conditions

were simultaneously assessed: the titre of the virus control and the D,, of the negative and positive sera were not statistically different from the mean of all the values previously obtained with the respective techniques.

3.2. FAVN test

To establish the specificity of the FAVN test, 414 sera from unvaccinated dogs were tested. Table 1

summanses D,, results according to the geographi- cal origin of dogs. For these sera, the D,, values ranged from 0.24 to 1.19 (mean D,, was less than 0.611. In fact, these data were overestimations con- sidering that most of the sera were available only in a small amount, and were therefore prediluted to l/3 prior to testing: in most cases, the fluorescence

was observed at the first dilution, and then it was only possible to establish the upper limit of the D,,. Converted in IU/ml, the mean titre was lower than

0.06 IU/ml. The highest D,, found (1.19) was significantly lower (p < 0.05) than the D,, threshold (1.52) previously determined for the reference serum.

Highly contaminated sera from 29 foxes obtained in rabies-free areas ranged from 0.24 to 1.19 (mean D,, = 0.57). According to these data, the specificity of the test was 100%. Precision was assessed by within-run CVs-9.9% (II = 8, mean D,, = 1.22, SD = 0.13) and 6.1% (n = 3, mean D,, = 1.65, SD = O.lO)-and by between-run CVs-5.4% (n = 3, mean D,, = 1.27, SD = 0.071, 7.41% (n = 3, mean D,, = 1.87, SD = 0.14) and 6.6% (n = 3, mean D,, = 1.65, SD = 0.18).

Accuracy was evaluated by titrating in three inde- pendent tests, known titres of the reference serum, and known dilutions of a dog serum. Serial dilutions near to 0.5 IU/ml of the reference serum were prepared in order to obtain suspensions titrating 0.2, 0.3, 0.4, 0.6, 0.7 and 1 IV/ml. Fig. 1 illustrates the

WHO standard reference serum

experimental titre

m/ml 2

1

r = 0.997 y = 0.89 X II=6 p < 0.001

ov I 1 1

D 0.5 1 1.5 2

expected titre NJ/ml

Diluted dog serum

experimental titre I&l *

I

0.02

r = 0.992

y = 0.88 x + 0.08 0.5

n=4

0.001 < -T 0.01 p

I ,

0 0.5 1 1.5 2

expected titre IU/ml

Fig. 1. Correlation between experimental and expected values for

the WHO standard reference serum and for a diluted serum. The

expected values were calculated knowing the dilution factors of

the sera and the titre of the undiluted sera. Each dilution was

titrated in three independent tests.

Table 2

F. C&pet et al. /Journal of Immunological Methods 2 12 ( 19%‘) 7947 83

Titration by the MNT and FAVN tests of samples from 20 unvaccinated and 30 vaccinated dogs

Result Number of sera % Agreement (disagreement) %

MNT FAVN test

Unvaccinated animals - 20

Vaccinated animals + + 16

- - 10

_ + 4

+ - 0

Among the vaccinees, 20 dogs produced low antibody levels (O-I IU/ml).

100 100

53.3 86.7

33.3

13.3 (13.3)

0

correlation between experimental and expected val-

ues for the two tested preparations. The correlation coefficients determined for the reference serum and

the diluted serum were 0.997 (p < 0.001) and 0.992 ( p < O.OOl), respectively.

3.3. Comparison between the FAVN test and the MNT

Table 2 compares the titres obtained by both MNT and the FAVN test on 30 and 20 sera from vaccinated and unvaccinated dogs, respectively. It should be noted that out of the 30 serum samples

from vaccinees, 20 were selected near 0.5 IU/ml (titres less than 1 W/ml), in an attempt to compare

the sensitivity of both techniques. The results ob- tained from the unvaccinated dogs using both me- thods were shown to correlate. Comparative titres obtained from vaccinees showed an agreement in most cases (86.7%). 4 sera with titres very near 0.5 IU/ml, were classified as negative by the MNT and

positive by the FAVN test, but no serum was found

to be positive by the MNT and negative by the FAVN test. This result suggests that the FAVN test is more sensitive than the MNT. Titres of the sera

obtained by both methods expressed in IU/ml showed a good correlation (n = 30, r = 0.82, p < 0.001).

3.4. Comparison between the FAVN test and the RFFIT

The FAVN test and the RFFIT were compared by

testing the same sera using both methods. Unvacci- nated (n = 78) and vaccinated (n = 77) animal sam-

ples were analysed. Among the sera sampled from vaccinees, 44 had low antibody levels (less than 1

IU/ml). The comparative titres are recorded in Table 3. Titres found by both methods were in close agree- ment (94.9% and 92.2% for unvaccinated and vacci- nated animals, respectively). However, 4 out of 78 negative samples (5.1%) tested by RFFIT provided a false positive result. with titres equal to 0.5 IU/ml.

Table 3 Titration by the RFFF and FAVN tests of 78 unvaccinated and 77 vaccinated dogs

Result Number of sera o/o Agreement (disagreement) %

RFFrIT FAVN test

Unvaccinated animals _ _ 14

+ _ 4

Vaccinated animals + + 47 _ _ 24

_ + 0

+ _ 6

Among the vaccinees, 44 dogs produced low antibody levels (O-1 IU/ml).

94.9

(5.1)

61.0 92.2

31.1

0 (7.8)

7.8

84 F. Cliquet et al. / Journal of Immunological Methods 212 (1998) 79-87

1.5 --

1.0

i

0.5 1

44 0 ’

l I’ 4

4 44 4

44

/

4, ,’ 4 ,* 44

4 44 ,I’

*#lb+

i

4

l * r/

1 i * r = 0.86

4 II=77

p -=z 0.001

---- x = Y

- y=l.l7x-0.13

95% confidence interval : l.O6<b< 1.27

1+--t- +-L-t + log 10

1.5 W/ml)

31 Ill/ml

RFFTT

Fig. 2. Correlation between log,, (IU/ml) of rabies antibody levels in the sera of vaccinated dogs titrated by RFFIT and FAVN test.

Probability density

25 m=0.32 SD= 0.14 FAVN test 2

1 m = 1.88 SD = 0.74

25 I RFFIT I

2

15

1 m = 0.88 SD = 0.22 m=1.86 SD=0.64

05

0 0 2 3

Fig. 3. Distribution of D,,, titres obtained using RFFIT and FAVN tests to assay samples from 78 unvaccinated and 77 vaccinated animals

F. Cliquet et al./Journal of Immunolo@zl Methods 212 (IUUXl 79-87 85

The comparative results from vaccinees showed that

6 out of 77 samples (7.8%) tested positive by the RFFIT (ranging from 0.5 to 0.7 IU/ml, geometric mean = 0.58, SD = 0.07) and negative by the FAVN test (ranging from 0.18 to 0.42 III/ml, geometric mean = 0.29, SD = 0.10). This result suggests either a lower sensitivity for the FAVN test compared to the RFFIT, or may indicate false positive responses with the RFFIT.

The correlation between the RFFIT and FAVN

titres found for the 77 vaccinees is illustrated in Fig. 2. Antibody titres, expressed in IU/ml, showed a

good correlation (n = 77, Y = 0.86, p < 0.001). The distributions of D,, values obtained with the

RFFIT and FAVN test for both the 78 negative

samples and the 77 samples from vaccinees were adjusted to give normal distributions (Fig. 3). The

D,, determined by the FAVN test on the unvacci- nated animals samples ranged from 0.25 to less than 1 (mean = 0.32, SD = 0.14), whereas the D,, deter- mined by the RFFIT on the same samples ranged from 0.25 to 1.32 (mean = 0.88, SD = 0.22). The

negative samples tested by the FAVN test showed significantly lower titres (p < 0.001) than the same samples analysed by RFFIT. For the vaccinees, the

means of all the values on the same samples were I .88 (SD = 0.74) according to the FAVN test, and 1.86 (SD = 0.64) according to the RFFIT. The dif-

ference between these two values was not statisti- cally significant. Furthermore, a comparison of the D,, distributions revealed that, using the FAVN test, the curves obtained with both unvaccinated and vac- cinated animal samples were clearly separated. On the other hand, using the RFFIT procedure, the

curves overlapped.

4. Discussion

The measurement of seroneutralising antibodies

has been commonly used for many years to assess the level of immunity to rabies in animals. It has been proposed as a measure that could replace or alleviate quarantine controls (Aubert, 1992). Testing the sera of properly vaccinated cats and dogs for the detection of rabies-specific antibodies is now cur- rently used by many European laboratories on a routine basis as part of the cross-border controls

(Klingebom, 1993). In order to assess the repro- ducibility of the RFFIT as practised by these labora- tories, an interlaboratory study on a panel of coded sera has been conducted and has revealed large discrepancies in the results obtained and numerous differences in the RFFiT methods used (report in progress). Most of the laboratories adapt the initial RFFIT as developed by Smith et al. (1973) and use microplates instead of Labtek chamber slides (as

proposed by Zalan et al., 1979); the initial descrip- tion of the standards employed when assessing fluo-

rescence was not sufficiently precise, and numerous read-out methods have been employed. The counting

of fluorescent fields (20 fields per Labtek chamber) is a protocol uniquely used for rabies antibody titra- tions. However, it is not very reliable for interlabora-

tory comparisons due to multiple sources of varia- tion Variations depending on the microscope used

(eyepiece, objective and magnification) are so large that the total number of fields per well may fluctuate. Furthermore, a bias in titre determination exists when reading the same field several times by mistake. To avoid these problems, the RFFIT practised in our

laboratory makes use of a global fluorescence read- ing, as described by Sureau et al. (1982). However, this is rather subjective and not reliable. A neutralisa- tion test has recently been published by Trimarchi et

al. (1996); this test uses different cell lines, different virus doses, and involves the reading of only one well per dilution.

The purpose of this study was to develop a test that would be easier to perform, and more reliable for the reading of the fluorescence. The FAVN test uses a qualitative microscopic reading (ccall-or-noth- ing>> reading), which is less time-consuming and not

subjective compared to methods that employ the counting of fields.

The specificity of the test evaluated by titrating more than 400 dog sera obtained from various areas

is 100%. Furthermore, contaminated sera from foxes sampled in unvaccinated areas have been success- fully analysed and were all found to be negative. When using the RFFIT, a cytotoxic effect may occur at low dilutions when the sera are contaminated (Smith et al., 1973; Kurz et al.. 1986: WHO, 1992); hence, there exists a risk of false positive titrations.

The data presented above, using sera with titres near 0.5 IU/ml, demonstrate that the FAVN test is

86 F. Cliquet et al. / Joumal of Immunological Methods 212 (19981 79-87

precise and accurate, when comparing the observed and expected values for diluted sera. The comparison of the FAVN test with both the MNT and the RFFIT was carried out by titrating negative and positive sera, including sera with titres near 0.5 III/ml. The results of the FAVN test agreed closely with those of the MNT (n = 30, r = 0.82, p < 0.001) and showed that the FAVN test is more sensitive than the MNT.

This last point has already been mentioned for the RFFIT when comparing it with the MNT (Smith et

al., 1973). The comparison of the FAVN and RFFIT tests has shown that the distribution of titres was

statistically identical for vaccinees whatever the test used. Correlation analyses on vaccinees showed that the FAVN test and the RFFIT correlated well (n = 77, r = 0.86, p < 0.001). Knowing that RFFIT can generate false positive titres (Smith et al., 1973; Kurz et al., 1986; WHO, 19921, we conclude that the

4 sera giving values of 0.5 III/ml by RFFIT and negative by the FAVN test were false positive re-

sponses. It appeared from this study that, for nega- tive samples, the FAVN test produced results that were significantly lower ( p < 0.001) than those of the RFFIT. In our hands, these results indicate that

the FAVN test provides a clear distinction between positive and naive antibody levels. This is not the case with the RFFIT, where the antibody levels overlap. In contrast, the FAVN test permits an effec- tive discrimination between naive and immunised dog sera, and it is possible to detect positive sera at a threshold lower than 0.5 IU/ml.

The numerous advantages of a test using mi- croplates compared with a test based on Labtek have

already been emphasized. The convenience of this all-or-nothing reading of the fluorescence is an addi-

tional advantage. The next step will be to automate this technique, for both the dilutions and additions of the products, and for the microscopic reading, per- mitting rapid testing of many samples. An interlabo- ratory collaborative assay involving international laboratories is in progress in order to assess the reproducibility of the test.

5. Conclusion

The FAVN test provides a satisfactory alternative to the RFFIT in the measurement of rabies antibod- ies in animal samples.

Acknowledgements

We are very grateful to Dr. Arthur King and Dr. Alex Wandeler for their suggestions and their inter- est in this study. We thank Mrs. Myriam Selve for

her excellent technical assistance.

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