Veterinary Microbiology, 31 (1992) 33-39 Elsevier Science Publishers B.V., Amsterdam

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Comparison of blocking dot ELISA and competitive ELISA, using a monoclonal antibody for detection ofbluetongue virus antibodies in cattle Ahmad Afshar, Gilles C. Dulac and Jos6 Riva Agriculture Canada, Animal Diseases Research Institute (Nepean), P.O. Box 11300, Station H, Nepean, Ontario, K2H 8P9, Canada (Accepted 18 September 1991 )

ABSTRACT Afshar, A., Dulac, G.C. and Riva, J., 1992. Comparison of blocking dot ELISA and competitive ELISA, using a monoclonal antibody for detection ofbluetongue virus antibodies in cattle, Vet. Microbiol., 31: 33-39. A blocking (B) dot enzyme-linked immunosorbent assay (ELISA), using a monoclonal antibody (mAb) against a group specific antigen of bluetongue virus (BTV) is described for the detection of BTV antibodies to BTV in cattle sera. Dots of BTV antigens were adsorbed to nitrocellulose (NC) strips and/or NC mounted in the windows of dipsticks. After blocking the remaining sites of the NC paper with milk powder solution and immersion in the test sample, the NC strips and dipsticks were exposed to mAb. Bound mAb was detected with peroxidase conjugated anti-mouse IgG (H and L). In the absence of anti-BTV antibody in the test sample, BTV antigen sites were reactive with mAb as indicated by a brown colored dot in the presence of the enzyme substrate, hydrogen peroxide and diaminobenzidine. In the presence of sufficient anti-BTV antibodies no color reaction was observed. The performance of these assays in detecting anti-BTV antibody in field blood eluate samples, prepared from whole blood dried on filter paper, from 395 bluetongue-free cattle in Canada and 635 sentinel cattle in Florida, USA, was evaluated and compared with the standard competitive (C) ELISA. The specificity of the dipstick B-dot ELISA was identical to that of the C-ELISA in testing of BT-free Canadian cattle but not in the testing of samples from the sentinel cattle in Florida, resulting in values of 100% diagnostic and 88.9% relative specificity, respectively. Based on the C-ELISA, the specificity of the NC strip B-dot ELISA was low and in the same order as that of the dipstick assay. The sensitivity of dipstick and NC strips assays in testing of 265 samples relative to C-ELISA results were 83.2% and 91.3%, respectively. The B-dot ELISA which is rapid, simple and inexpensive may be an alternative field test for screening and monitoring cattle, and possibly other ruminants, exposed to BTV for control and surveillance programs.

INTRODUCTION

The diagnosis ofbluetongue virus (BTV) exposure, generally a sub-clinical disease in cattle, relies primarily on assays for detection of antibodies to group specific antigen (Jochim, 1985; Parsonson, 1990). While the agar gel immu0378-1135/92/$05.00

© 1992 Elsevier Science Publishers B.V. All rights reserved.

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nodiffusion (AGID) test remains the most widely used assay for detection of group specific antibodies to BTV, concerns about its diagnostic specificity has led to the development of several monoclonal antibody (mAb)-based enzyme immunosorbent assays (ELISA). Anderson (1984) and Lunt et al. ( 1988 ) described a blocking (B) ELISA in which the immobilized BTV antigen was first reacted with the test serum and then with a group specific murine mAb to BTV. Anti-BTV antibody if present in the test serum, blocked the antigen preventing its reaction with the mAb in the second step of the test. Afshar et al. (1987a, 1989) and House et al. (1990) reported mAb-based competitive (C) ELISAs in which the test serum and the mAb reacted simultaneously to compete for BTV antigen. While the microplate-based ELISA tests are highly sensitive, specific and quantitative, they are not applicable at the field level because they require sophisticated equipment and expertise. Based on the microtiter plate B-ELISA procedure of Anderson (1984), Afshar et al. (1987b) described a simple and inexpensive B-dot ELISA in which nitrocellulose (NC) paper strips were used as solid support for BTV antigen to screen bovine and ovine sera for antibodies to BTV. After exposure to the test serum the NC strips were reacted with the mAb and subsequently with peroxidase conjugated anti-mouse immunoglobulin-G (IgG). A negative reaction for a test serum without detectable anti-BTV antibody was easily visualized as a dot after enzyme degradation of a precipitating chromogenic substrate. In the presence of antibodies to BTV, similar to the B-ELISA, the BTV antigen sites, represented in the dot in NC strips, were blocked and the mAb and subsequently the enzyme-labeled antiglobulin can not bind and consequently no color (dot) developed. The performance of the B-dot ELISA on a limited number of sera, collected from cattle and sheep infected experimentally with BTV, was reported to be better than the AGID (Afshar et al., 1987b). Recently, Gupta et al. (1990) described a dot i m m u n o b i n d i n g assay (DIA) based on an indirect (I) ELISA procedure originally reported for serodiagnosis of bluetongue by Manning and Chen (1980). It was found that DIA was superior to the microplate-based I-ELISA when serum samples from naturally infected sheep were tested. The present report describes a simple and rapid B-dot ELISA for detection of antibodies to BTV in cattle. The test makes use of NC in form of strips or dipsticks as solid-phase support for immobilization of BTV antigen. In addition, a study comparing the B-dot ELISA to the C-ELISA was carried out, using eluates from whole blood, collected and dried on filter paper, as test samples. MATERIAL AND METHODS

BTV antigens Lots of BTV antigen for C-ELISA were prepared from baby hamster kidney cell line (BHK-21 ) according to a m e t h o d described previously (Anderson,

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1984; Afshar et al., 1987b). The BTV antigen used for the B-dot ELISA was a cell culture fluid stock (AA15-85 ) with a titer of 105 plaque forming units ( P F U ) of BTV per ml and its corresponding control negative tissue culture fluid.

Blood samples Whole blood samples were collected in 1988 from 395 cattle slaughtered at Abattoir du Nord, Montr6al, Que. Canada (bluetongue-free) and in 1989 from 635 sentinel cattle in Florida (bluetongue-endemic), USA (supplied by Drs. P. Gibbs, P. Nicolletti and T. Tuekam, College of Veterinary Medicine, University of Florida). Blood samples were allowed to diffuse into and saturate a part of 90-mm diameter filter paper disks (Whatman). The filters were allowed to dry at ambient temperature and disks of 6 m m diameter were punched and stored in screw cap vials at - 2 0 °C until used. Each small disk retained approximately 5/tl of blood and the required dilution of blood eluate was made by soaking the appropriate number of disks in 0.01M phosphate buffer saline, pH 7.4 (PBS) containing 0.05% Tween 20 (PBST). The blood eluate was used for B-dot and C-ELISA after storage overnight at 4°C and gentle shaking at room temperature (rt) for 10 min.

B-dot ELISA procedure The B-dot ELISA was a modification of the procedure previously described (Afshar et al., 1987b). Following optimization studies, 1/tl of both the BTVinfected and control-cell culture fluids were applied separately to one end of NC strips (2.5 × 0.5 cm) or to the two NC windows located at the tip of the dipstick (Cerex Corporation, Gaithersburg, MD, USA). After drying the NC strips and the dipsticks at 37 °C for 5 min, a second application of BTV and control cell culture antigen was made and allowed to dry for 25 min at 37°C. The NC strips and the dipsticks were immersed for 30 min at rt in PBS containing 3% skim milk powder (Carnation, blocking buffer) for 30 min at rt. Prior to this step, the dipstick wells were filled with the blocking buffer, using a Pasteur pipette. This precaution prevented the trapping of air bubbles over the NC windows. The NC strips and the dipsticks were rinsed and washed at least three times in PBS prior to their exposure to blood eluate sample, at a final dilution of 1:4 in PBST. While in the next step the NC strips were immersed into diluted blood eluate sample, the dipstick wells were filled with the appropriate test sample, and all incubated at rt in a h u m i d chamber for 60 min. The NC strips and the dipsticks were washed in several changes of PBST and then they were incubated with a predetermined dilution ( 1 : 50) of BTV mAb 3-17-A3 (supplied by Dr. J. Anderson, Pirbright Laboratory, Woking, England, U K ) in PBST for 30 rain at rt. Following another washing cycle with PBST to remove u n b o u n d mAb, the NC strips and dipsticks were exposed for 30 min at rt to a 1 : 1000 dilution in PBST of peroxidase conjugated

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goat anti-mouse IgG (Bethyl Laboratory, Montgomery, TX, USA). After a final wash in PBS the NC strips and the dipsticks were immersed in diaminobenzidin (DAB) (Sigma, St. Louis, MO, USA) and H202 substrate as described previously (Afshar et al., 1986). After 3 min the enzymatic reaction was stopped by washing the NC strips and the dipsticks in tap water and air drying prior to visual inspection for colour development. Positive (BTV type 10 antiserum) and negative bovine serum samples were included in each test run for quality control purposes. C-ELISA The C-ELISA test was carried out as previously described (Afshar et al., 1989 ), except that blood eluates were used instead of serum samples and the peroxidase labeled goat anti-mouse IgG used at 1 : 500 dilution. Samples were considered positive if they inhibited 50% or more of the mAb activity. RESULTS

The results of the screening B-dot ELISA, using NC strips and the standard C-ELISA for the blood eluate samples from sentinel cattle in Florida, USA, are shown in Table 1. Relative to the C-ELISA results, the sensitivity of the B-dot ELISA was slightly low ( 97.1% ) and the specificity was low (62.6%). Of the total 635 samples 12 and 84 resulted in false negative and false positive reactions, respectively (Table 1 ). None of the 395 samples from Eastern Canada, an area free of bluetongue, were positive in the B-clot ELISA, using dipsticks and C-ELISA. In contrast, when 265 available blood eluate samples from Florida were tested by dipstick ELISA discrepant results were recorded TABLE1 Comparison of the blocking (B) dot ELISA, using nitrocellulose strips, and the competitive ( C ) ELISA in the detection of antibody to bluetongue virus in 635 blood eluate samples collected from sentinel cattle in Florida B-dot ELISA

Competitive ELISA Positive

Relative sensitivity I

Relative specificity 2

Overall agreement 3

(%)

(%)

+ve

-ve

(%)

398

12

97.1

84

141

84.9 Negative

62.6

t Sensitivity = (B-dot ELISA and C-ELISA p o s i t i v e s / C - E L I S A positives) X 100 2Specificity = (B-dot ELISA and C-ELISA negatives/C-ELISA negatives) X 100 3Overall agreement= (B-dot ELISA and C-ELISA positives)+ (B-dot ELISA and C-ELISA negatives ) / (C-ELISA positives + C-ELISA negatives )

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COMPARISON OF BLOCKING DOT ELISA AND COMPETITIVE ELISA

TABLE 2 Comparison of the blocking (B) dot ELISA tests and competitive (C) ELISA in the detection of antibody to bluetongue virus in 265 blood eluate samples collected from sentinel cattle in Florida Nitrocellulose strips

Dipsticks

Number ofsera

Number ofsera

+ C-ELISA Positive 178 Negative 7

Relative sens ~ Relative spec 2 (%) (%)

-

17 63

91.3 90

+

-

154 9

31 71

Relative Sens j (%)

Relative Spec 2 (%)

83.2 88.9

~Sensitivity = (B-dot ELISA and C-ELISA positives/C-ELISA positives) × 100 2Specificity= (B-dot ELISA and C-ELISA negatives/C-ELISA negatives) × 100

(Table 2). A total of 40 samples resulted in false negative (31) and false positive (9) reactions giving a sensitivity of 83.2% and specificity of 88.9% relative to the C-ELISA results (Table 2). Comparison of the B-dot ELISA, using NC strips and dipsticks relative to the C-ELISA for the 265 Florida blood eluate samples as presented in Table 2, demonstrated a better overall performance for the B-dot ELISA using NC strips. Relative to the C-ELISA results there were 16 less false reactions for the NC strips test than the dipstick B-dot ELISA (Table 2 ). DISCUSSION

The C-ELISA test (Afshar et al., 1989 ) which was previously validated for its specificity and sensitivity in detecting group-specific antibodies to BTV, was the reference test for this study. Based on a B-ELISA procedure (Anderson, 1984) we previously developed a simple dot ELISA, using NC membrane as solid phase, to test a limited n u m b e r of animal sera for antibodies to BTV (Afshar et al., 1987b). In the present study, we have extended the application of the B-dot ELISA to the testing of several hundreds of bovine field samples. In order to simulate field conditions, where equipment for refrigeration and separation of serum from the blood samples are often not available, we used whole blood dried on filter paper as test sample. The availability of dipsticks with NC windows/wells has also prompted us to use them as reactant cartier in the B-dot ELISA for detecting antibodies to BTV in blood eluate samples, simulating field conditions. Unlike the NC strips, the handling of dipsticks throughout the steps of the dot ELISA was easier and did not require tedious manipulations to avoid tear and loss of the NC strips. Dipsticks have been used successfully to develop ELISAs for the detection of various anti-

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body/antigen complexes (Nielsen et al., 1985; Pappas 1986; Gupta et al., 1990). With the 395 samples from Canadian cattle, the dipstick B-dot ELISA performance was similar to that of the C-ELISA where no antibodies to BTV were demonstrable by both assays. We have also reported similar test performance by the dot ELISA on NC strips for other Canadian cattle (Afshar et al., 1987b). The B-dot ELISA appears to have potential application as a specific, rapid, practical and economical field test for monitoring cattle free from bluetongue antibody. Nitrocellulose strip B-dot ELISA results on 635 blood eluate samples from sentinel cattle, Florida (Table 1 ) compared favorably with the C-ELISA sensitivity but not its specificity. In contrast to the Canadian samples, there were far too many samples ( 84 ) classified as false positive, requiring confirmatory testing by C-ELISA. Such a rate of false positives, relative to C-ELISA results, makes the NC strip B-dot ELISA as non-specific a test as the agar gel immunodiffusion (AGID) assay that we reported for a limited number of bovine field sera (65) collected from Oklahoma (Afshar et al., 1987b). Furthermore, we found similar specificity for the dipstick B-dot ELISA compared with that of the NC strip B-dot ELISA when both tests are compared with the C-ELISA results for 265 blood eluate samples that were of sufficient volume for performing all the three assays (Table 2 ). The diagnostic specificity of the dipstick B-dot ELISA may be higher than that suggested by these results of relative specificity and must be confirmed by more extensive field trials for different animal populations. Given a suitable diagnostic specificity, the dipstick B-dot ELISA may be an alternative field screening test for monitoring animal m o v e m e n t and surveillance programs in bluetongue free countries. The whole blood samples collected and dried on filter paper has an application for disease surveillance among domestic and possibly wild ruminants during the hunting season. ACKNOWLEDGEMENTS

We are indebted to Professors Paul Gibbs and Paul Nicolletti of Department of Infectious Diseases, College of Veterinary Medicine, University of Florida and Dr. T. Tuekam from Cameroon for the supply of blood samples from Florida sentinel herds.

REFERENCES Afshar, A., Wright, P.F. and Dulac, G.C., 1986. Dot-enzyme immunoassay for visual detection of antibodies to pseudorabies virus in swine serum. J. Clin. Microbiol., 23: 563-567. Afshar, A., Thomas, F.C., Wright, P.F., Shapiro, J.L., Shettigara, P.T. and Anderson, J., 1987a. Comparison of competitive and indirect enzyme-linked immunosorbent assays for detection

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of bluetongue virus antibodies in serum and whole blood. J. Clin. Microbiol., 25: 17051710. Afshar, A., Thomas, F.C., Wright, P.F., Shapiro, J.L., Anderson, J. and Fulton, R.W., 1987b. Blocking dot-ELISA, using a monoclonal antibody for detection of antibodies to bluetongue virus in bovine and ovine sera. J. Virol. Methods, 18:271-280. Afshar, A., Thomas, F.C., Wright, P.F., Shapiro, J.L. and Anderson, J., 1989. Comparison of competitive ELISA, indirect ELISA and standard AGID tests for detecting bluetongue virus antibodies in cattle and sheep. Vet. Rec., 124:136-141. Anderson, J., 1984. Use of monoclonal antibody in a blocking ELISA to detect group specific antibodies to bluetongue virus. J. Immunol. Methods, 74:139-149. Gupta, Y., Chand, P., Singh, A. and Jain, N.C., 1990. Dot immunobinding assay in comparison with enzyme-linked immunosorbent assay for the detection of bluetonge virus antibodies in sheep. Vet. Microbiol., 22:365-371. House, C., House, J.A. and Beminger, M.L., 1990. Detection of bluetongue group-specific antibody by competitive ELISA. J. Vet. Diagn. Invest., 2:137-139. Jochim, M.M., 1985. An overview of diagnostics for bluetongue. In : T.L. Barber and M.M. Jochim (Editors), Bluetongue and related orbiviruses. Alan R. Liss. New York, NY. pp. 423-433. Lunt, R.A., White, J.R. and Blacksell, S.D., 1988. Evaluation ofa monoclonal antibody blocking ELISA for the detection of group specific antibodies to bluetongue virus in experimental and field sera. J. Gen. Virol., 69: 2729-2740. Manning, J.S. and Chen, M.F., 1980. Bluetongue virus: Detection ofantiviral immunoglobulin by means of enzyme-linked immunosorbent assay. Curr. Microbiol., 4:381- 385. Nielsen, K., Ballinger, R., Stiller, J. and Rosenbaum, B., 1985. A "dipstick" ELISA for detection of antibody to Brucella abortus in cattle sera. Can. J. Comp. Med., 49: 298-302. Pappas, M.G., 1986. Rapid serodiagnosis of parasitic infections by dot ELISA using "Dipsticks". Trans. R. Soc. Trop. Med. Hyg., 80: 1006. Parsonson, I.M., 1990. Pathology and pathogenesis ofbluetongue infection. In: P. Roy and B.M. Gorman (Editors), Bluetongue viruses. Curr. Top. Microbiol. Immunol., Vol. 162:119141.

Comparison of blocking dot ELISA and competitive ELISA, using a monoclonal antibody for detection of bluetongue virus antibodies in cattle.

A blocking (B) dot enzyme-linked immunosorbent assay (ELISA), using a monoclonal antibody (mAb) against a group specific antigen of bluetongue virus (...
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