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Epidemiological evaluation of a monoclonal ELISA detecting bovine viral diarrhoea pestivirus antigens in field blood samples of persistently infected cattle
85
Jorrmd of. Virological ~~~t~[~~s,40 ( 1992) 85 -94 1“ 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00 VIRMET 01399
Epidemiological evaluation of a monoclonal ELISA detecting bovine viral diarrhoea pestivirus antigens in field blood samples of persistently infected cattle B. Mignon,
S. Waxweiler,
E. Thiry, D. Boulanger, P.-P. Pastoret
J. Dubuisson
and
Summary An enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies for capture and detection, was developed for detecting bovine viral diarrhoea virus (BVDV) antigens in blood samples. The test was evaluated using 761 field samples of known status (viraemic or not). When an appropriate cut-off value was chosen, the sensitivity, speci~city, and predictive values of the assay were lOO%, higher than the values obtained by classical virus isolation. Correlation with the latter technique exceeded 90%. The ELISA is a good candidate for replacing virus isolation as a reference method for BVDV antigen detection in persistently infected carriers. A method based on the mean of the standard deviation ratio can be used to choose the cut-off value in order to optimise reproducibility. ELISA: Bovine viral diarrhoea virus; Monoclonal infected cattle; BVDV, antigen detection
antibody;
BVDV, persistently
Introduction Bovine viral diarrhoea virus (BVDV) is the pestivirus which causes bovine viral diarrhoea and mucosal disease in cattle (Brownlie, 1990). When a c~~~r~,sp(~n~ie~~.e to: B. Mignon, Department of Virology. Faculty of Veterinary Medicine. University of Liege. B-4000 Sart Tilman-Liege, Belgium.
X6
pregnant cow is infected with a non-cytopathic BVDV strain, it may produce a persistently infected calf that appears healthy but has persistent viraemia and constantly excretes large alnounts of virus (Brownlie et al., 1987). Persistent excretors are responsible for maintaining BVDV within herds and are liable to succumb to mucosal disease. Although the prevalence of persistently infected cattle is usually less than 1% (Meyling, 1984), economic losses due to BVDV, especially to persistent infection (Bennet and Done, 1986), make detection of carriers essential. Classical detection methods are time-consuming and expensive, requiring virus amplification in monolayers of cultured susceptible cells and detection by indirect methods, such as indirect immunofluorescence (Waxweiler et al., 1989). We have modified slightly the monoclonal enzyme-linked immunosorbent assay (ELISA) described by Mignon et al. (1991) and applied it in the field. This permitted the evaluation of its validity as a diagnostic tool and its advantages as compared to virus isolation (VI) for detection of asymptomatic carriers.
Materials
and Methods
Between January and July 1991, duplicate IO-ml heparinised blood samples were collected from all 6-month-old steers introduced to the Centre de Selection Bovine de Ciney and from cattle from private farms, whose veterinarians had requested screening for BVDV infection. One thousand and six samples were thus made available for VI screening. As some samples had been collected from the same animals at various intervals in order to confirm presumed persistent viraemia, a total of 989 animals were, in fact, tested. 761 of the 1006 samples were tested by ELISA. ELBA Antigen was prepared according to the method of Fenton et al. (1990), as modified by Mignon et al. (1991). Briefly, IO-ml heparinised blood samples were centrifuged for 30 min at 1000 x g to obtain white blood cells. Lysis of remaining red cells with ammonium chloride and one wash with PBS yielded the final leucocyte pellet. The latter was incubated for 10 min at room temperature with 300 ~1 of Nonidet P-40 and centrifuged for 15 min at 10 000 x g. The supernatant was stored at -80°C and used as antigen. The monoclonal ELISA for assaying BVDV antigen in persistently infected cattle (Mignon et al., 1991) was modified slightly. Microtiter plates (Nunc) were coated overnight at 4°C with capture-purified monoclonal antibodies (mAbs) (200 ng of each mAb per well) (Table 1). Plates were washed five times, as between each of the subsequent steps, with PBS containing 0.02% Tween 80.
87
TABLE 1 Properties
of mAbs used in sandwich ELISA
Antibody”
(Glyco)protein
Capture mAbs NY8 NY 15 as 22
&SO
Detecting mAbs NY6 OS 23
48 12Oj80
speciticityb @Da)
48
“Capture and detecting mAbs were chosen according to relative affinity, reciprocal competition (Mignon et al., 1991) and reactivity spectrum against several BVDV strains (Boulanger et al., 1991). bDetermined by radioimmunoprecipitation (Boulanger et al., 1991).
Antigen was incubated for 1 h at 37°C. A mixture of two biotinylated puri~ed mAbs (Table I) was then added (100 ng of each mAb per well) and incubated for 1 h at room temperature. This was followed by incubation with biotinylated streptavidin-horseradish peroxidase (Amersham), diluted l/400, for 1 h at 37°C. Substrate (ABTS, Boehringer, Mannheim) was incubated for 8 h at room temperature and the absorbance (A4i4) was measured with a Titertek Multiscan, using a 414 nm filter. Throughout the procedure, 100-/d reagent volumes were used. Virus isolation Detection of BVDV by indirect immunofluorescence was carried out as described by Waxweiler et al. (1989). Briefly, Madin Darby bovine kidney (MDBK) cells were infected with white blood cells from centrifuged (1000 x g for l/2 h) and frozen (at -8OC) samples. After 1 week of incubation, supernatants from frozen and thawed infected cell cultures were used to infect MDBK cefls grown in 96-well plates. After 3 days, cells were fixed for 45 min at -20°C in acetone/distilled water (955, v/v) and stained by an indirect immunofluorescence test using a cocktail of anti-BVDV mAbs and fluorosceinlabelled anti-mouse rabbit Igs (Prosan). Virus isolation and the ELISA were carried out independently by two individuals. Evaluation of the ELISA The objective classification of blood samples as viraemic (V+) or not viraemic (V-) was done by virus isolation. The latter was performed in our laboratory and in another laboratory using fetal bovine kidney cells. ‘Home’ VI was considered the standard test and compared to the new ELISA. A set of I51 randomly chosen V- samples and all 26 V + samples available
XX
(from 16 different animals) were simultaneously tested by ELISA using two immunoplates. Contingency tables were constructed in order to evaluate the intrinsic validity of the ELISA by calculating the sensitivity (SC) and specificity (@) of the test for different cut-off values. Bayes’s theorem (Jenicek and Cleroux. 1983) was used to determine the predictive values (PI’+ and PI’-, respectively) of positive and negative ELISA-results for different prevalence 01) values. The Sr, Sp and PV values of the ELISA and VI test were then compared. Correlation between the two tests was measured by determining the kappa (1~) ratio (Martin et al., 1987). The standard deviation ratio (SDR) method: described by Parkinson at al. (1988), was used to interpret the ELISA data. This method was assessed as a means of avoiding reproducibility problems. In SDR analysis, briefly. the mean and the standard deviation of the negative samples, constituting the bulk of the population. are used to decide which samples are truly positive; each plate assay undergoes its own statistical analysis, which generates an appropriate cut-off.
Results Among the 1006 samples tested by the 3-step procedure, 41 were V + These corresponded to 24 different animals, so the prevalence (I,) of BVDV viraemia was in fact 2.4%. Table 2 shows the contingency tables constructed with ELISA results for different cut-off values of the absorbance at 414 nm (A4i4). TABLE
2
Classification
of ELISA
results”
v+
v --
26 -1” Ih 0
,2 1” I39 lU
3X I39
26
0 ‘I’ 151 -IV I51
26 151 171
25 1.1’ , Fh
15C; ::
26
ISI
Cut-off at 0.3” ELISA + ELISA ~
Cut-off at 0.4’ ELISA t ELISA -
Cut-off at 0.5” ELISA + ELISA ~
TP. true positive; FN, false negative; FP, false positive: TN. true negative. “A sample with an absorbance higher than the chosen cut-off value is classified ‘SC,. 100%: Sp. 92.1% CsP, 100%: Sp. lOO”,G. “SC. 96.2%: sp. 100%
25 I57 I77
as positive.
89
100 90 -
//
80 -
/
70 -
I
60-
/’
/’
5040-
/
Se -----
r’
SP
I’
30-i 20
I 0,30
0,20
1 0,40
I 0,50 A414
Fig.
1,Sensitivity (Se = TP/(TP+
FN)) and specificity (Sp = TN/(TN off values. The test is considered positive when A 414 is greater
+ FP)) of the ELISA at different than the chosen cut-off value.
cut-
Se and Sp calculated from contingency tables were both 100% when the cut-off was chosen between 0.4 and 0.45 (Fig. 1, Table 2). The probability (PI’+) that a positive result in ELISA was truly V+ equalled loo%, for cut-off values above 0.4. At cut-off values equal to or lower than 0.4, PI’+ was clearly p-dependent, as shown in Fig. 2. The predictive value of a negative result (PV= (1 -p)Sp/{(l -p) Sp +p( 1 -Se)}) was always above 99% for cut-offs between 0.2 and 0.55 and p values inferior to 5%. Table 3 shows the lower Se and PV+ of the ‘home’ VI, as compared to the ELISA (Figs. 1 and 2). The correlation between the two methods was good, however (K > 0.4) for an ELBA cut-off between 0.25 and 0.55. The correlation was excellent (K > 0.9) for a cut-off at 0.35 or higher, as could be predicted from the Se, Sp (Fig. l), and PI’ values (Fig. 2) of the ELISA.
TABLE
3
Classification
of ‘home’
VI + VI-
VI results V+
V-
32 TP * t+
3 963 TN 966
FP
40 See Table 2 for legend. Se, 80%; Sp, 99.7%; PV@=O.l%).
>99%
(5%
0.1%);
PV+
varying
35 971 I006
from 93.1%
(p=5%)
to 20.5%
90 100
(%I “+
80 --m---
60
A414=0.25 A414~0.3 A41 4~0.35
40 20
0 0
1
2
3
4
5
6
Pt*hl Fig. 2. Predictive value of a positive result (PP’+ = p.Sei{p. Se+ (1 -a)(1 -S&j) in ELISA at varloub cut off values and a&different values of BVDV viraemia prevalence (J).
Two immunoplates were used simultaneously to test V + and V - samples. The mean and standard deviation of the absorbance for Vsamples were, respectively, 0.21 and 0.032 on plate 1 and 0.248 and 0.042 on plate 2, indicating that the choice of an appropriate cut-off could be affected by reproducibility of the ELISA. For this reason, the AdI cut-off values were calculated by the SDR method for each plate individually, the values chosen being 0.361 for plate I and 0.457 for plate 2. Under these conditions, Se and SIP were both 100%.
Discussion Our previous monoclonal ELISA (Mignon et al., 1991) was modified slightly and tested on 761 field blood samples. Epidemiological analysis, including determination of Se, Sp, PP’ values and of the correlation with classical VI, demonstrated the applicability of the ELISA in the field as a reference method for AVDV antigen detection in persistently infected cattle. The mAbs were chosen for their high relative affinity, the absence of competition between them (Mignon et al., 1991), and their broad reactivity against BVDV strains (Boulanger et al., 1991). By combining mAbs raised against the highly conserved non-structural 80 kDa protein (Donis and Dubovi, 1987; Collett et al., 1989) and the 48 kDa glycopxotein {one of the two major glycoproteins), it was assumed that false negatives would be fewer or
91
eliminated altogether. Our results confirm this assumption: for a cut-off at 0.45 or lower, the sensitivity value of the ELISA was lOO%, while the specificity value remained 90% or higher for a cut-off above 0.3. The sensitivity and specificity of the ELISA were determined from a sample containing 15 1 V and 26 V+ samples, the latter being derived from seven farms and from the Centre de Selection Bovine de Ciney, thus covering a range of pestivirus isolates. Sampling was not representative of the whole bovine population, where p is well below 14.7% (26/177), often totalling 0.5 to 1% (Waxweiler et al., 1989). Determination of Se and SJJ values, however, does not require that the prevalence be the same among the samples as in the general population; in our experience, increasing the proportion of V+ samples provided more stringency for determining accurate SC and Sp values. Considering the epidemiological role of persistently viraemic cattle in maintaining BVDV circulation in the herd and the usually low prevalence of infection, the foremost intrinsic quality to be sought in a detection test is high sensitivity, that is, avoidance of false negatives. Of course, the specificity must be acceptable for economical reasons: re-testing too many animals, eliminating healthy ones, are to be avoided. The ELISA, therefore, with its higher sensitivity and specificity than classical virus isolation, appears appropriate as a reference method for detecting BVDV viraemia. This conclusion is supported by the high PV+ and P1/- values, even at low prevalence, when the discriminating cut-off is 0.4 to 0.45. In our experience, this is cut-off range to be used. Furthermore, the excellent correlation between VI and the ELISA indicates that the cheaper, easier ELISA could replace, with advantage, the VI technique. Antigen for the ELISA was prepared from buffy coat, for which BVDV has a known affinity (Bielefeldt-Ohmann et al., 1987). NP-40-treated whole blood is obtained more quickly and was used successfully for the ELISA carried out with anti-48-kDa mAbs (results not shown), but it produced too much background with anti-120 kDa/80 kDa mAbs. The latter were used however because of the high conservation of the 80-kDa protein. Our ELISA differs markedly from the ELISAs described by Fenton et al. (1990) and Shannon et al. (1991), who used a polyclonal antiserum for antigen detection (Fenton) or capture (Shannon). The potency of mAbs and their advantage as compared to polyclonal antibodies are that they can be obtained easily and indefinitely, with constant quality, and in large quantities. Variations in the measured A4i4 for V+ samples (data not shown) may be due to the mAbs having different relative affinities for different viral strains, but another cause could be variable quantities of antigen in samples. For this reason, Waxweiler et al. (1989) recommend screening for persistent viraemia in cattle over 6 months of age, when maternal antibodies can no longer interfere with VI. Of interest is our preliminary finding that potential vaccinal antipestivirus antibodies did not prevent antigen detection by the ELISA. Indeed, four animals were diagnosed as persistent carriers although they had been vaccinated 6 months before testing. Our aim was to validate the ELISA with samples known to be either V+ or
V - . When applying the test to samples of unknown status, it will be necessary to compare the results with positive and negative controls. One positive control per plate will prove that the test has been correctly performed. Negative controls can be samples on the plate. since the majority of the tests should be negative and are more representative of the population being tested than a value determined for a stored specimen. This approach provides an additional criterion for choosing the cut-off absorbance: reproducibility of the ELISA. Our experience indicates that the variability of the absorbance values is, rather linked to the ELISA technique and to the technician him~herself than to the samples. Differences can even be recorded between plates. sometimes due to washing steps (see the differences in the means of V- AJl3 values on two simultaneously used plates). Thus, a certain lack of reproducibility can occur, especially if negative controls are taken on the plate, but this problem can be minimized if the cut-off is determined by the SDR method (Parkinson et al.. 1988), since each plate assay undergoes its own statistical analysis, which generates the appropriate cut-off. This facilitates introduction of the ELISA into other laboratories, which can reevaluate the optimum cut-off under their own conditions. We have thus developed a means of detecting BVBV antigen in cattle by an ELISA using monoclo~~al antibodies for capture and detection. This is the first reported evaluation of such an ELISA on field material and in which epidemiological parameters have been determined. This analysis has demonstrated the qualities and the applicability of the EL.ISA. compared to classical virus isolation, notwithstanding the excellent correlation between the two methods. The ELISA could be very useful in sanitary programmes aimed at detecting persistently BVDV infected cattle, a major challenge in controlling bovine viral diseases.
Acknowledgements
We would like to thank M. L,oncar for excellent technical assistance. D. Vandenbergh (Eurogentec sa) for testing samples by virus isolation and the ‘Centre de Selection Bovine de Ciney’. This work was supported by grants from the ‘Institut pour l-encouragement de la Recherche Scientifique dans I’lndustrie et I’Agricutture’ (IRSIA) and Rhhne-Mkrieux Company. Dr. J. Dubuisson is a Senior Research Assistant of the ‘Ponds National Beige de la Recherche Scientifique’ (FNRS).
References Bennet. social
Biclefcldt
K.M.
and Dono,
cost-benefit
Ohmann,
5.7’. (1986) Control 01‘thc bovine pestivirus syndrome in cattle: ;L USC I’or Proc. Sot. Vet. Epid. Prevent. Med.. Thrusfield. Edimbourg. f-1.. Ronxholt, 1.. and Rloch. B. (lYX7) llmonstration of bovine wral diarrhoca analysis’?
93 virus in peripheral blood mononuclear cells of persistently infected, clinically normal cattle. J. Gen. Virol. 68, 1971--1982. Boulanger, D., Waxweiler, S., Karelle, L., Loncar, M., Mignon, B.. Dubuisson. J., Thiry, E. and Pastorct, P.P. (1991) Characterization of monodonal antibodies to bovine viral diarrhoea virus: evidence of a neutralizing activity against gp48 in the prcscncc of goat anti-mouse immunoglobulin serum. J. Gen. Virol. 72, 1195 I lY8. Brownlie, J. (1990) Pathogen&s of mucosal disease and molecular aspects of bovine virus diarrhoea virus. Vet. Microbial. 23. 371-382. Brownlie, J.. Clarke, M.C., Howard, C.J. and Pocock, D.H. (19X7) Pathogenesis and epidemiology of bovine virus diarrhoea virus infection of‘ cattle. Ann. Rcch. Vet. 128, 157 166. Collet, MS.. Moennig. V. and Horzinck M.C. (1989) Recent advances in Pestivirus research. J. Gen. Viral. 70, 253 266. Donis, R.Q. and Dubovi, E.J. (1987) Differences in virus-induced polypeptid~s in cells infected by cytopathic and n(~n-cytopathic biotypes of bovine virus diarrhoea-mucosal disease virus. Virology 158, I68 173. Fenton. A., Entrican, G., Herring. J.A. and Nettleton, P.F. (1990) An ELISA for detecting pestivirus antigen in the blood of sheep persistently infected with border disease virus. J. Viral. Methods 27. 253 260. Jenicek, M. and Cleroux, R. (1983) Epidemiologic. Principcs. Techniques. Apphcdtions. Edisem Inc.. Ste Hyacinte, Quebec. Martin, SW.. Meek. A.H. and Willeberg, P. (1987) Vctcrinary Epidemiology. Principles and methods. Iowa State University Press. Mcyling, A. (1984) Detection of the BVD virus in viraemic cattle by an indirect immunoperoxidase technique. In: M.S. McNulty and J.B. McFerran (Eds), Recent Advances in Virus diagnosis. Martinus Nijhoff, Boston, pp. 37 46. Mignon, B., Dubuisson, J., Baranowski, E., Koromyslov, I., Ernst, E.. Boulangcr, D., Waxweiler, S. and Pastoret P.P. (1991) A monoclonal ELISA for bovine diarrhoea pestivirus antigen dotcction in persistently infected cattle. J. Virol. Methods 35, 177 18X. Parkinson. R.M., Conradie, L.V., Milner, L.V. and Marimuthu, T. (1988) The interpretation of ELISA results by means of the standard deviation ratio. J. Immunol. Methods 115, 105.. 110. Shannon. A.D.. Richards, S.G., Kirkland, P.D. and Moylc A. (1991) An antigen-capture ELISA detects pestivirus antigens in blood and tissues of immunotolerant carrier cattle. J. Virol. Methods 34, I 12. Waxweiier. S.. Karelle-Bui-Thi, L.. Sneyers, M., Lambert, A.F., Delchambre, M., Dubuisson, J., Thiry, E.. Antoine, H., Dive. M., Detal, G. and Pastoret P.P. (1989) Circulation de souchcs noncytopathogenes du virus BVD-MD dans des lots de taurillons. Ann. Med. Vet. 133, 68 I -690.
Jorrmd of. Virological ~~~t~[~~s,40 ( 1992) 85 -94 1“ 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00 VIRMET 01399
Epidemiological evaluation of a monoclonal ELISA detecting bovine viral diarrhoea pestivirus antigens in field blood samples of persistently infected cattle B. Mignon,
S. Waxweiler,
E. Thiry, D. Boulanger, P.-P. Pastoret
J. Dubuisson
and
Summary An enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies for capture and detection, was developed for detecting bovine viral diarrhoea virus (BVDV) antigens in blood samples. The test was evaluated using 761 field samples of known status (viraemic or not). When an appropriate cut-off value was chosen, the sensitivity, speci~city, and predictive values of the assay were lOO%, higher than the values obtained by classical virus isolation. Correlation with the latter technique exceeded 90%. The ELISA is a good candidate for replacing virus isolation as a reference method for BVDV antigen detection in persistently infected carriers. A method based on the mean of the standard deviation ratio can be used to choose the cut-off value in order to optimise reproducibility. ELISA: Bovine viral diarrhoea virus; Monoclonal infected cattle; BVDV, antigen detection
antibody;
BVDV, persistently
Introduction Bovine viral diarrhoea virus (BVDV) is the pestivirus which causes bovine viral diarrhoea and mucosal disease in cattle (Brownlie, 1990). When a c~~~r~,sp(~n~ie~~.e to: B. Mignon, Department of Virology. Faculty of Veterinary Medicine. University of Liege. B-4000 Sart Tilman-Liege, Belgium.
X6
pregnant cow is infected with a non-cytopathic BVDV strain, it may produce a persistently infected calf that appears healthy but has persistent viraemia and constantly excretes large alnounts of virus (Brownlie et al., 1987). Persistent excretors are responsible for maintaining BVDV within herds and are liable to succumb to mucosal disease. Although the prevalence of persistently infected cattle is usually less than 1% (Meyling, 1984), economic losses due to BVDV, especially to persistent infection (Bennet and Done, 1986), make detection of carriers essential. Classical detection methods are time-consuming and expensive, requiring virus amplification in monolayers of cultured susceptible cells and detection by indirect methods, such as indirect immunofluorescence (Waxweiler et al., 1989). We have modified slightly the monoclonal enzyme-linked immunosorbent assay (ELISA) described by Mignon et al. (1991) and applied it in the field. This permitted the evaluation of its validity as a diagnostic tool and its advantages as compared to virus isolation (VI) for detection of asymptomatic carriers.
Materials
and Methods
Between January and July 1991, duplicate IO-ml heparinised blood samples were collected from all 6-month-old steers introduced to the Centre de Selection Bovine de Ciney and from cattle from private farms, whose veterinarians had requested screening for BVDV infection. One thousand and six samples were thus made available for VI screening. As some samples had been collected from the same animals at various intervals in order to confirm presumed persistent viraemia, a total of 989 animals were, in fact, tested. 761 of the 1006 samples were tested by ELISA. ELBA Antigen was prepared according to the method of Fenton et al. (1990), as modified by Mignon et al. (1991). Briefly, IO-ml heparinised blood samples were centrifuged for 30 min at 1000 x g to obtain white blood cells. Lysis of remaining red cells with ammonium chloride and one wash with PBS yielded the final leucocyte pellet. The latter was incubated for 10 min at room temperature with 300 ~1 of Nonidet P-40 and centrifuged for 15 min at 10 000 x g. The supernatant was stored at -80°C and used as antigen. The monoclonal ELISA for assaying BVDV antigen in persistently infected cattle (Mignon et al., 1991) was modified slightly. Microtiter plates (Nunc) were coated overnight at 4°C with capture-purified monoclonal antibodies (mAbs) (200 ng of each mAb per well) (Table 1). Plates were washed five times, as between each of the subsequent steps, with PBS containing 0.02% Tween 80.
87
TABLE 1 Properties
of mAbs used in sandwich ELISA
Antibody”
(Glyco)protein
Capture mAbs NY8 NY 15 as 22
&SO
Detecting mAbs NY6 OS 23
48 12Oj80
speciticityb @Da)
48
“Capture and detecting mAbs were chosen according to relative affinity, reciprocal competition (Mignon et al., 1991) and reactivity spectrum against several BVDV strains (Boulanger et al., 1991). bDetermined by radioimmunoprecipitation (Boulanger et al., 1991).
Antigen was incubated for 1 h at 37°C. A mixture of two biotinylated puri~ed mAbs (Table I) was then added (100 ng of each mAb per well) and incubated for 1 h at room temperature. This was followed by incubation with biotinylated streptavidin-horseradish peroxidase (Amersham), diluted l/400, for 1 h at 37°C. Substrate (ABTS, Boehringer, Mannheim) was incubated for 8 h at room temperature and the absorbance (A4i4) was measured with a Titertek Multiscan, using a 414 nm filter. Throughout the procedure, 100-/d reagent volumes were used. Virus isolation Detection of BVDV by indirect immunofluorescence was carried out as described by Waxweiler et al. (1989). Briefly, Madin Darby bovine kidney (MDBK) cells were infected with white blood cells from centrifuged (1000 x g for l/2 h) and frozen (at -8OC) samples. After 1 week of incubation, supernatants from frozen and thawed infected cell cultures were used to infect MDBK cefls grown in 96-well plates. After 3 days, cells were fixed for 45 min at -20°C in acetone/distilled water (955, v/v) and stained by an indirect immunofluorescence test using a cocktail of anti-BVDV mAbs and fluorosceinlabelled anti-mouse rabbit Igs (Prosan). Virus isolation and the ELISA were carried out independently by two individuals. Evaluation of the ELISA The objective classification of blood samples as viraemic (V+) or not viraemic (V-) was done by virus isolation. The latter was performed in our laboratory and in another laboratory using fetal bovine kidney cells. ‘Home’ VI was considered the standard test and compared to the new ELISA. A set of I51 randomly chosen V- samples and all 26 V + samples available
XX
(from 16 different animals) were simultaneously tested by ELISA using two immunoplates. Contingency tables were constructed in order to evaluate the intrinsic validity of the ELISA by calculating the sensitivity (SC) and specificity (@) of the test for different cut-off values. Bayes’s theorem (Jenicek and Cleroux. 1983) was used to determine the predictive values (PI’+ and PI’-, respectively) of positive and negative ELISA-results for different prevalence 01) values. The Sr, Sp and PV values of the ELISA and VI test were then compared. Correlation between the two tests was measured by determining the kappa (1~) ratio (Martin et al., 1987). The standard deviation ratio (SDR) method: described by Parkinson at al. (1988), was used to interpret the ELISA data. This method was assessed as a means of avoiding reproducibility problems. In SDR analysis, briefly. the mean and the standard deviation of the negative samples, constituting the bulk of the population. are used to decide which samples are truly positive; each plate assay undergoes its own statistical analysis, which generates an appropriate cut-off.
Results Among the 1006 samples tested by the 3-step procedure, 41 were V + These corresponded to 24 different animals, so the prevalence (I,) of BVDV viraemia was in fact 2.4%. Table 2 shows the contingency tables constructed with ELISA results for different cut-off values of the absorbance at 414 nm (A4i4). TABLE
2
Classification
of ELISA
results”
v+
v --
26 -1” Ih 0
,2 1” I39 lU
3X I39
26
0 ‘I’ 151 -IV I51
26 151 171
25 1.1’ , Fh
15C; ::
26
ISI
Cut-off at 0.3” ELISA + ELISA ~
Cut-off at 0.4’ ELISA t ELISA -
Cut-off at 0.5” ELISA + ELISA ~
TP. true positive; FN, false negative; FP, false positive: TN. true negative. “A sample with an absorbance higher than the chosen cut-off value is classified ‘SC,. 100%: Sp. 92.1% CsP, 100%: Sp. lOO”,G. “SC. 96.2%: sp. 100%
25 I57 I77
as positive.
89
100 90 -
//
80 -
/
70 -
I
60-
/’
/’
5040-
/
Se -----
r’
SP
I’
30-i 20
I 0,30
0,20
1 0,40
I 0,50 A414
Fig.
1,Sensitivity (Se = TP/(TP+
FN)) and specificity (Sp = TN/(TN off values. The test is considered positive when A 414 is greater
+ FP)) of the ELISA at different than the chosen cut-off value.
cut-
Se and Sp calculated from contingency tables were both 100% when the cut-off was chosen between 0.4 and 0.45 (Fig. 1, Table 2). The probability (PI’+) that a positive result in ELISA was truly V+ equalled loo%, for cut-off values above 0.4. At cut-off values equal to or lower than 0.4, PI’+ was clearly p-dependent, as shown in Fig. 2. The predictive value of a negative result (PV= (1 -p)Sp/{(l -p) Sp +p( 1 -Se)}) was always above 99% for cut-offs between 0.2 and 0.55 and p values inferior to 5%. Table 3 shows the lower Se and PV+ of the ‘home’ VI, as compared to the ELISA (Figs. 1 and 2). The correlation between the two methods was good, however (K > 0.4) for an ELBA cut-off between 0.25 and 0.55. The correlation was excellent (K > 0.9) for a cut-off at 0.35 or higher, as could be predicted from the Se, Sp (Fig. l), and PI’ values (Fig. 2) of the ELISA.
TABLE
3
Classification
of ‘home’
VI + VI-
VI results V+
V-
32 TP * t+
3 963 TN 966
FP
40 See Table 2 for legend. Se, 80%; Sp, 99.7%; PV@=O.l%).
>99%
(5%
0.1%);
PV+
varying
35 971 I006
from 93.1%
(p=5%)
to 20.5%
90 100
(%I “+
80 --m---
60
A414=0.25 A414~0.3 A41 4~0.35
40 20
0 0
1
2
3
4
5
6
Pt*hl Fig. 2. Predictive value of a positive result (PP’+ = p.Sei{p. Se+ (1 -a)(1 -S&j) in ELISA at varloub cut off values and a&different values of BVDV viraemia prevalence (J).
Two immunoplates were used simultaneously to test V + and V - samples. The mean and standard deviation of the absorbance for Vsamples were, respectively, 0.21 and 0.032 on plate 1 and 0.248 and 0.042 on plate 2, indicating that the choice of an appropriate cut-off could be affected by reproducibility of the ELISA. For this reason, the AdI cut-off values were calculated by the SDR method for each plate individually, the values chosen being 0.361 for plate I and 0.457 for plate 2. Under these conditions, Se and SIP were both 100%.
Discussion Our previous monoclonal ELISA (Mignon et al., 1991) was modified slightly and tested on 761 field blood samples. Epidemiological analysis, including determination of Se, Sp, PP’ values and of the correlation with classical VI, demonstrated the applicability of the ELISA in the field as a reference method for AVDV antigen detection in persistently infected cattle. The mAbs were chosen for their high relative affinity, the absence of competition between them (Mignon et al., 1991), and their broad reactivity against BVDV strains (Boulanger et al., 1991). By combining mAbs raised against the highly conserved non-structural 80 kDa protein (Donis and Dubovi, 1987; Collett et al., 1989) and the 48 kDa glycopxotein {one of the two major glycoproteins), it was assumed that false negatives would be fewer or
91
eliminated altogether. Our results confirm this assumption: for a cut-off at 0.45 or lower, the sensitivity value of the ELISA was lOO%, while the specificity value remained 90% or higher for a cut-off above 0.3. The sensitivity and specificity of the ELISA were determined from a sample containing 15 1 V and 26 V+ samples, the latter being derived from seven farms and from the Centre de Selection Bovine de Ciney, thus covering a range of pestivirus isolates. Sampling was not representative of the whole bovine population, where p is well below 14.7% (26/177), often totalling 0.5 to 1% (Waxweiler et al., 1989). Determination of Se and SJJ values, however, does not require that the prevalence be the same among the samples as in the general population; in our experience, increasing the proportion of V+ samples provided more stringency for determining accurate SC and Sp values. Considering the epidemiological role of persistently viraemic cattle in maintaining BVDV circulation in the herd and the usually low prevalence of infection, the foremost intrinsic quality to be sought in a detection test is high sensitivity, that is, avoidance of false negatives. Of course, the specificity must be acceptable for economical reasons: re-testing too many animals, eliminating healthy ones, are to be avoided. The ELISA, therefore, with its higher sensitivity and specificity than classical virus isolation, appears appropriate as a reference method for detecting BVDV viraemia. This conclusion is supported by the high PV+ and P1/- values, even at low prevalence, when the discriminating cut-off is 0.4 to 0.45. In our experience, this is cut-off range to be used. Furthermore, the excellent correlation between VI and the ELISA indicates that the cheaper, easier ELISA could replace, with advantage, the VI technique. Antigen for the ELISA was prepared from buffy coat, for which BVDV has a known affinity (Bielefeldt-Ohmann et al., 1987). NP-40-treated whole blood is obtained more quickly and was used successfully for the ELISA carried out with anti-48-kDa mAbs (results not shown), but it produced too much background with anti-120 kDa/80 kDa mAbs. The latter were used however because of the high conservation of the 80-kDa protein. Our ELISA differs markedly from the ELISAs described by Fenton et al. (1990) and Shannon et al. (1991), who used a polyclonal antiserum for antigen detection (Fenton) or capture (Shannon). The potency of mAbs and their advantage as compared to polyclonal antibodies are that they can be obtained easily and indefinitely, with constant quality, and in large quantities. Variations in the measured A4i4 for V+ samples (data not shown) may be due to the mAbs having different relative affinities for different viral strains, but another cause could be variable quantities of antigen in samples. For this reason, Waxweiler et al. (1989) recommend screening for persistent viraemia in cattle over 6 months of age, when maternal antibodies can no longer interfere with VI. Of interest is our preliminary finding that potential vaccinal antipestivirus antibodies did not prevent antigen detection by the ELISA. Indeed, four animals were diagnosed as persistent carriers although they had been vaccinated 6 months before testing. Our aim was to validate the ELISA with samples known to be either V+ or
V - . When applying the test to samples of unknown status, it will be necessary to compare the results with positive and negative controls. One positive control per plate will prove that the test has been correctly performed. Negative controls can be samples on the plate. since the majority of the tests should be negative and are more representative of the population being tested than a value determined for a stored specimen. This approach provides an additional criterion for choosing the cut-off absorbance: reproducibility of the ELISA. Our experience indicates that the variability of the absorbance values is, rather linked to the ELISA technique and to the technician him~herself than to the samples. Differences can even be recorded between plates. sometimes due to washing steps (see the differences in the means of V- AJl3 values on two simultaneously used plates). Thus, a certain lack of reproducibility can occur, especially if negative controls are taken on the plate, but this problem can be minimized if the cut-off is determined by the SDR method (Parkinson et al.. 1988), since each plate assay undergoes its own statistical analysis, which generates the appropriate cut-off. This facilitates introduction of the ELISA into other laboratories, which can reevaluate the optimum cut-off under their own conditions. We have thus developed a means of detecting BVBV antigen in cattle by an ELISA using monoclo~~al antibodies for capture and detection. This is the first reported evaluation of such an ELISA on field material and in which epidemiological parameters have been determined. This analysis has demonstrated the qualities and the applicability of the EL.ISA. compared to classical virus isolation, notwithstanding the excellent correlation between the two methods. The ELISA could be very useful in sanitary programmes aimed at detecting persistently BVDV infected cattle, a major challenge in controlling bovine viral diseases.
Acknowledgements
We would like to thank M. L,oncar for excellent technical assistance. D. Vandenbergh (Eurogentec sa) for testing samples by virus isolation and the ‘Centre de Selection Bovine de Ciney’. This work was supported by grants from the ‘Institut pour l-encouragement de la Recherche Scientifique dans I’lndustrie et I’Agricutture’ (IRSIA) and Rhhne-Mkrieux Company. Dr. J. Dubuisson is a Senior Research Assistant of the ‘Ponds National Beige de la Recherche Scientifique’ (FNRS).
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