J Vet Diagn Invest 3:144-147 (1991)
A competitive ELISA for detection of antibodies to the group antigen of bluetongue virus J. J. Reddington, G. M. Reddington, N. J. MacLachlan Abstract. A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect antibodies to the group antigen of bluetongue virus (BTV). The epitope recognized by the BTV-specific monoclonal antibody was confirmed, by immunofluorescence staining of monolayers of virus-infected Vero cells, to be present on BTV serotypes 2, 10, 11, 13, and 17 but not on epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2. Sera from BTV-inoculated ruminants and rabbits were used to evaluate the cELISA and to compare its specificity and sensitivity with that of the conventional BTV-specific agar gel immunodiffusion (AGID) and serum neutralization (SN) tests. Rabbit antisera to the 5 serotypes of BTV present in the United States had cELISA titers (inverse of the final dilution of serum that gave > 20% inhibition) that ranged from 32 to > 1,024. Seroconversion of the 8 calves and lambs inoculated with BTV was detected by all 3 serologic tests (SN, AGID, cELISA) by 6 weeks after inoculation. Specificity of the cELISA test was confirmed with bovine sera that contained neutralizing antibodies to EHDV but not to the 5 serotypes of BTV present in the United States; these sera gave positive results by AGID test but were negative by The sensitivity and specificity of test was further confirmed by analysis of a panel of bovine test sera supplied by the National the is a superior test for detection of BTV groupVeterinary Services Laboratories, indicating that the specific antibodies in sera from ruminants in the United States.
Bluetongue virus (BTV) is a member of the Reoviridae and is the prototype virus of the genus Orbivirus.4 The virus is transmitted between susceptible ruminants, both wild and domestic, by biting gnats? Some BTV-infected ruminants develop severe disease characterized by pyrexia, leukopenia, oral erosions, facial edema, and hyperemia of the oral mucous membranes and coronary band; bluetongue disease usually occurs in sheep and wild ruminants in the United States. 5 Although the BTV infection of cattle is usually asymptomatic, cattle are considered a reservoir host of BTV. Viremia in infected cattle is prolonged, providing virus for insect transmission to more susceptible ruminant species . 5 Because bluetongue disease is a continuing threat to ruminants in areas of the world in which BTV infection does not occur and because cattle are considered a reservoir host of BTV, countries free of BTV infection frequently ban or restrict the importation of ruminants and their genetic products (embryos and semen) from BTV-endemic areas of the world, such as the United States. 13 Such trade restrictions reflect in part the insensitivity of the agar gel immunodiffusion (AGID) test. Although a wide variety of tests will
detect BTV-specific antibodies, the AGID test has been used most extensively in the United States for detection of BTV serogroup-specific antibodies. It has become increasingly obvious that the AGID is neither sufficiently specific nor sensitive to justify its continued use as the standard regulatory test for serologic detection of previous BTV infection of ruminants? False negative reactions with the AGID test occur because precipitating antibodies often are slow to become detectable and persist for variable periods after exposure of ruminants to BTV; 6 , 1 2 false positive reactions may occur as a consequence of exposure of ruminants to non-BTV orbiviruses such as epizootic hemorrhagic disease virus (EHDV)? Several different blocking or competitive enzymelinked immunosorbent assays (cELISA) for detection of serum antibodies directed to BTV group-specific antigens have been recently described. 1-3,9,11 T h e is both more sensitive and more specific than the AGID test and is increasingly being used for serologic diagnosis of BTV infection. The purpose of this report is to describe a similar cELISAa using the 290 monoclonal antibody (MAb). Materials and methods
From Consultants for Applied Biosciences, Inc., Wilton, CT 06897 (Reddington, Reddington), and the Department of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616 (MacLachlan). Received for publication September 7, 1990.
viruses. Prototype strains of BTV serotypes 2, 10, 11, 13, and 17 and EHDV serotypes 1 and 2b were plaque-purified in agar-overlaid African green monkey kidney (Vero) cells, and stock cultures were propagated in baby hamster kidney (BHK-21) cells. 144
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Table 1. Titers of bluetongue virus (BTV)-specific antibody in BTV-specific MAb. The production and preliminary charhyperimmune rabbit antisera as determined by competitive enzymeacterization of an MAb, identified as 290, specific for core protein VP7 of BTV serotype 10 has been described previ- linked immunosorbent assay (cELISA). l5 ously. The MAb was biotinylated according to the manufacturer’s instructions.c Presence of the epitope recognized by this MAb on each of the 5 serotypes of BTV and 2 serotypes of EHDV was determined by direct immunofluorescence staining of BTV-infected Vero cells. Monolayers of Vero cells grown on chamber slides were infected with each serotype of BTV or EHDV and fixed in 80% acetone after approximately 24 hr. Cells were then incubated with the biotinylated MAb, followed by fluorescein isothiocyanateconjugated avidin.d Polyclonal antisera. Antisera were raised against BTV in rabbits, colostrum-deprived calves, and lambs. Individual rabbits were immunized against each of the 5 different BTV serotypes; rabbits were intravenously inoculated with gradient purified BTV, followed by subcutaneous inoculation with BTV-infected suckling mouse brain resuspended in The negative control serum utilized in the test was a pooled Freund’s adjuvant.8 Colostrum-deprived calves were reared sample from colostrum-deprived isolation-reared calves. in insect-secure isolation facilities and were intravenously Other serologic tests. The BTV-SN test was done in 96, inoculated with approximately 1 x 106 tissue culture infec- well plates as previously described.8 Titers are expressed as tive doses (TCID50) of BTV; 2 calves were inoculated with the inverse of the final dilution of serum that provided at serotype 10, and single calves were inoculated with serotypes least 50% protection of BHK-21 cell monolayers against 250 11, 13, and 17. Two additional calves were inoculated with TCID 50 of virus. The AGID test was performed according uninfected lysate of BHK-21 cells. Three lambs seronegative to standard protocol,i and results were reported as either to BTV, as determined by serum neutralization (SN) test and positive or negative. immune precipitation assay, 14 were raised in insect-secure isolation facilities and intravenously inoculated with 5 x 10 4 Results TCID 50 of BTV serotype 10. An additional lamb was inocThe epitope recognized by the MAb used in the ulated with a lysate of uninfected BHK-21 cells. Calves were cELISA was confirmed by direct immunofluorescence 1-20 wk old at inoculation, and lambs were approximately staining of virus-infected Vero cells to be present on 10 wk old. all 5 serotypes of BTV found in the United States but A panel of 30 bovine test sera was obtained and evaluated with the cELISA. Included in this panel were sera positive not on serotype 1 or 2 of EHDV. Hyperimmune rabbit and negative by BTV-AGID test and sera that contained antisera to each of the 5 serotypes of BTV inhibited neutralizing antibodies to EHDV serotypes 1 or 2 but not to binding (20%) of the MAb in the cELISA at final diBTV. An additional serum sample from a bull was also ob- lutions that ranged from 1:32, with antisera to serotype tained;f this serum gave a positive result with the BTV-AGID 13, to greater than 1: 1,024 with the antisera to serotest but contained only neutralizing antibody to EHDV and types 10, 11, and 17 (Table 1). The control rabbit not to BTV. antisera did not inhibit binding of the MAb at a diCompetitive ELISA test. The procedure used for the lution of 1:2. cELISA was a modification of a previously described methRuminant sera were used to compare the sensitivity od.2 ELISA plates were coated with a lysate of BTV serotype and specificity of the cELISA with those of the SN and lo-infected BHK-21 cells. At 36 hr after infection, the inAGID tests (Table 2). False positive results were not fected cells were subjected to a freeze/thaw cycle and diluted in 0.1 M NaHCO3 buffer. This antigen preparation was then obtained with any of the 3 serologic tests using sera used to coat round-bottom microtiter plates.g Test sera were from the control calves and lamb. Results obtained diluted in dilution buffer and added to antigen-coated wells with antisera from lambs inoculated with BTV seroprior to the addition of the biotinylated BTV-specific MAb. type 10 indicate that all 3 tests specifically identified After incubation for 1 hr, wells were washed and horseradish infected animals by 2 weeks after inoculation; howperoxidase-conjugated streptavidin was added and incubated ever, the cELISA appeared to be more sensitive than for 30 min. 2,2' Azino-bis(3-ethylbenzthiazoline-6-sulfonic either the AGID or SN tests. Serum obtained from acid (ABTS) substrate was added at a concentration of 0.22 lamb 3 at 7 days after inoculation, for instance, conmg/ml, and the reaction was stopped with sodium dodecyl sulfate (SDS) after approximately 10 min of incubation. End- tained neither precipitating nor neutralizing antibodies point optical density (OD) readings were made on a plate but gave a high degree of inhibition (77%) as deterreader h at 410 nm. Results obtained with the cELISA are mined by cELISA. Serum obtained from lamb 2 at 1 expressed as percent inhibition, as determined by the follow- week after inoculation was negative by the AGID test, whereas the same sample had a relatively high cELISA ing formula:
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Reddington, Reddington, MacLachlan Table 2. Comparison of serologic tests for bluetongue virus (BTV)-specific antibodies in sera from inoculated calves and lambs.
titer (39% inhibition). All 3 serologic tests also specif- BTV. An additional bovine serum that was BTV-AGID ically identified BTV-specific antibodies in sera from test positive, BTV-SN test negative, and EHDV-SN calves inoculated with each of the 4 different serotypes test positive also was determined to be cELISA test of BTV; all 5 infected calves were seropositive by the negative. These data strongly suggest that the AGID 3 tests at 2-6 weeks after inoculation. Sensitivity of test results from these 3 bovine sera are false positive the 3 tests varied among calves and with the serotype reactions and that the results of the cELISA and SN of BTV used. For instance, serum taken from calf 1 at tests are more valid. 4 weeks after inoculation had a high cELISA titer (58% inhibition) and a high SN titer (64) but was negative Discussion by the AGID test. Furthermore, serum taken from this Although the AGID test has served as the standard calf at 6 weeks after inoculation gave only a weak positive reaction with the AGID test, whereas the cELISA regulatory test for serologic diagnosis of BTV infection titer was high (78% inhibition). In contrast, serum taken in the United States and much of the rest of the world, from calf 4 (which was inoculated with BTV serotype it is increasingly obvious that the test is neither suffi13) at 4 weeks after inoculation was positive by the ciently sensitive nor specific to justify its continued 2,3,6 AGID test and had a high SN titer (64) but had a low use Our results confirm that the cELISA test is both more sensitive and more specific than the AGID test, (19% inhibition) titer as determined by cELISA. The cELISA was further evaluated using a panel of which is to be expected because the cELISA test quancattle sera obtained from the National Animal Disease titates only the displacement of BTV-specific MAb by Center. The cELISA confirmed results of the AGID the test serum. Problems with epitopes common to 10 test with 2 exceptions; both these sera were positive •BTV and closely related serogroups that produce false by the AGID test, negative by cELISA, and both sera positive reactions with the AGID test are thus avoided. contained neutralizing antibodies to EHDV but not to In addition, the cELISA is quantitative and avoids
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subjective interpretation, which is inherent in the interpretation of the AGID test. Results of this study indicate that sensitivity of the cELISA is comparable to that of the SN test; the SN test is generally accepted as the most sensitive and specific of the available tests for serologic detection of BTV infection of ruminants. The SN test is too cumbersome and laborious for routine use in the diagnostic laboratory, especially when sera are to be screened for the presence of antibodies to multiple serotypes of BTV. Comparison of the results of this study with data obtained previously with some of the same sera also indicate that the as presented here has comparable sensitivity to that of the Western immunoblot and immune precipitation assays.14 Immunoblotting and especially immune precipitation are highly sensitive and specific methods of assaying for the presence of viral-specific polypeptides, but, like the SN test, they are not appropriate for routine diagnostic use. Any used for serologic diagnosis of BTV in the United States obviously must incorporate an MAb directed at an epitope present on all serotypes of BTV found in the United States. In addition, the epitope recognized by the MAb must be unique to BTV serogroup viruses. Results of this and prior studies clearly indicate that the VP7-specific 290 is well suited for use in the VP7 is a component of the core of the BTV virion and is considered a group-specific protein, 7 which is compatible with our observation that the epitope recognized by our VP7-specific is present on all serotypes of BTV found in the United States. Bluetongue virus-infected cattle and sheep mount a strong humoral immune response to VP7;14 therefore, a that detects antibodies specific for VP7 is a logical test for serologic confirmation of BTV infection of ruminants. In summary, the is a superior test for serologic diagnosis of BTV infection of ruminants in the United States because it has comparable sensitivity to the SN test, requires significantly less time to run, and provides objective results. Sources and manufacturers a. BluePlate Special, Consultants for Applied Biosciences, Wilton, CT. b. Supplied by Dr. J. L. Stott, School of Veterinary Medicine, University of California, Davis, CA. c. Pierce, Rockford, IL. d. Zymed Laboratories, South San Francisco, CA. e. United States Department of Agriculture, National Veterinary Services Laboratory, Ames, IA.
f. Supplied by Dr. B. I. Osbum, School of Veterinary Medicine, University of California, Davis, CA. g. Falcon Microtest III, Becton Dickinson and Co., Lincoln Park, NJ. h. MR 5000, Dynatech Laboratories, Chantilly, VA. i. California Veterinary Diagnostic Laboratory, Davis, CA.
References 1. 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. 2. Ashfar A, Thomas FC, Wright PF, et al.: 1987, Comparison of competitive and indirect enzyme-linked immunosorbent assays for detection of bluetongue virus antibodies in serum and whole blood. J Clin Microbiol 25: 1705-1710. 3. Ashfar A, Thomas FC, Wright PF, et al.: 1989, Comparison of competitive ELISA, indirect ELISA, and standard AGID tests for detecting bluetongue virus antibodies in cattle and sheep. Vet Ret 124:136-141. 4. Borden EC, Shope RE, Murphy FA: 1971, Physiochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus-a new taxonomic group: physiochemical and serological studies. J Gen Virol 13:262-271. 5. Bowne JG: 1971, Bluetongue disease. Adv Vet Sci Comp Med 15: 1-46. 6. Della-Porta AJ, Parsonson IM, McPhee DA: 1985, Problems in the interpretation of diagnostic tests due to cross-reactions between orbiviruses and broad serological responses in animals. Prog Clin Biol Res 178:445-454. 7. French TJ, Roy P: 1990, Synthesis of bluetongue virus (BTV) core-like particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. J Virol 64: 15301536. 8. Heidner HW, MacLachlan NJ, Fuller FJ, et al.: 1988, Bluetongue virus genome remains stable during prolonged infection of cattle. J Gen Virol 69:2629-2636. 9. House C, House JA, Beminger ML: 1990, Detection of bluetongue group-specific antibody by competitive ELISA. J Vet Diagn Invest 2: 137-139. 10. Huismans H, Bremer CW, Barber TL: 1979, The nucleic acid and proteins of epizootic haemorrhagic disease virus. Onderstepoort J Vet Res 46:95-104. 11. Lunt RA, White JR, Blacksell SD: 1988, Evaluation of a 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. 12. MacLachlan NJ, Schore CE, Osbum BI: 1984, Antiviral responses of bluetongue virus inoculated bovine fetuses and their dams. Am J Vet Res 45:1469-1473. 13. Osbum BI: 1988, Economics of bluetongue in the United States. Proc 4th Symp Arbovirus Res Aust, pp. 245-247. 14. Richards RG, MacLachlan NJ, Heidner HW, et al.: 1988, Comparison of virologic and serologic responses of lambs and calves infected with bluetongue virus serotype 10. Vet Microbiol 18: 233-242. 15. Whetter LE, MacLachlan NJ, Gebhard DH, et al.: 1989, Bluetongue virus infection of bovine monocytes. J Gen Virol 70: 1663-1676.
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A competitive ELISA for detection of antibodies to the group antigen of bluetongue virus J. J. Reddington, G. M. Reddington, N. J. MacLachlan Abstract. A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect antibodies to the group antigen of bluetongue virus (BTV). The epitope recognized by the BTV-specific monoclonal antibody was confirmed, by immunofluorescence staining of monolayers of virus-infected Vero cells, to be present on BTV serotypes 2, 10, 11, 13, and 17 but not on epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2. Sera from BTV-inoculated ruminants and rabbits were used to evaluate the cELISA and to compare its specificity and sensitivity with that of the conventional BTV-specific agar gel immunodiffusion (AGID) and serum neutralization (SN) tests. Rabbit antisera to the 5 serotypes of BTV present in the United States had cELISA titers (inverse of the final dilution of serum that gave > 20% inhibition) that ranged from 32 to > 1,024. Seroconversion of the 8 calves and lambs inoculated with BTV was detected by all 3 serologic tests (SN, AGID, cELISA) by 6 weeks after inoculation. Specificity of the cELISA test was confirmed with bovine sera that contained neutralizing antibodies to EHDV but not to the 5 serotypes of BTV present in the United States; these sera gave positive results by AGID test but were negative by The sensitivity and specificity of test was further confirmed by analysis of a panel of bovine test sera supplied by the National the is a superior test for detection of BTV groupVeterinary Services Laboratories, indicating that the specific antibodies in sera from ruminants in the United States.
Bluetongue virus (BTV) is a member of the Reoviridae and is the prototype virus of the genus Orbivirus.4 The virus is transmitted between susceptible ruminants, both wild and domestic, by biting gnats? Some BTV-infected ruminants develop severe disease characterized by pyrexia, leukopenia, oral erosions, facial edema, and hyperemia of the oral mucous membranes and coronary band; bluetongue disease usually occurs in sheep and wild ruminants in the United States. 5 Although the BTV infection of cattle is usually asymptomatic, cattle are considered a reservoir host of BTV. Viremia in infected cattle is prolonged, providing virus for insect transmission to more susceptible ruminant species . 5 Because bluetongue disease is a continuing threat to ruminants in areas of the world in which BTV infection does not occur and because cattle are considered a reservoir host of BTV, countries free of BTV infection frequently ban or restrict the importation of ruminants and their genetic products (embryos and semen) from BTV-endemic areas of the world, such as the United States. 13 Such trade restrictions reflect in part the insensitivity of the agar gel immunodiffusion (AGID) test. Although a wide variety of tests will
detect BTV-specific antibodies, the AGID test has been used most extensively in the United States for detection of BTV serogroup-specific antibodies. It has become increasingly obvious that the AGID is neither sufficiently specific nor sensitive to justify its continued use as the standard regulatory test for serologic detection of previous BTV infection of ruminants? False negative reactions with the AGID test occur because precipitating antibodies often are slow to become detectable and persist for variable periods after exposure of ruminants to BTV; 6 , 1 2 false positive reactions may occur as a consequence of exposure of ruminants to non-BTV orbiviruses such as epizootic hemorrhagic disease virus (EHDV)? Several different blocking or competitive enzymelinked immunosorbent assays (cELISA) for detection of serum antibodies directed to BTV group-specific antigens have been recently described. 1-3,9,11 T h e is both more sensitive and more specific than the AGID test and is increasingly being used for serologic diagnosis of BTV infection. The purpose of this report is to describe a similar cELISAa using the 290 monoclonal antibody (MAb). Materials and methods
From Consultants for Applied Biosciences, Inc., Wilton, CT 06897 (Reddington, Reddington), and the Department of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616 (MacLachlan). Received for publication September 7, 1990.
viruses. Prototype strains of BTV serotypes 2, 10, 11, 13, and 17 and EHDV serotypes 1 and 2b were plaque-purified in agar-overlaid African green monkey kidney (Vero) cells, and stock cultures were propagated in baby hamster kidney (BHK-21) cells. 144
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Table 1. Titers of bluetongue virus (BTV)-specific antibody in BTV-specific MAb. The production and preliminary charhyperimmune rabbit antisera as determined by competitive enzymeacterization of an MAb, identified as 290, specific for core protein VP7 of BTV serotype 10 has been described previ- linked immunosorbent assay (cELISA). l5 ously. The MAb was biotinylated according to the manufacturer’s instructions.c Presence of the epitope recognized by this MAb on each of the 5 serotypes of BTV and 2 serotypes of EHDV was determined by direct immunofluorescence staining of BTV-infected Vero cells. Monolayers of Vero cells grown on chamber slides were infected with each serotype of BTV or EHDV and fixed in 80% acetone after approximately 24 hr. Cells were then incubated with the biotinylated MAb, followed by fluorescein isothiocyanateconjugated avidin.d Polyclonal antisera. Antisera were raised against BTV in rabbits, colostrum-deprived calves, and lambs. Individual rabbits were immunized against each of the 5 different BTV serotypes; rabbits were intravenously inoculated with gradient purified BTV, followed by subcutaneous inoculation with BTV-infected suckling mouse brain resuspended in The negative control serum utilized in the test was a pooled Freund’s adjuvant.8 Colostrum-deprived calves were reared sample from colostrum-deprived isolation-reared calves. in insect-secure isolation facilities and were intravenously Other serologic tests. The BTV-SN test was done in 96, inoculated with approximately 1 x 106 tissue culture infec- well plates as previously described.8 Titers are expressed as tive doses (TCID50) of BTV; 2 calves were inoculated with the inverse of the final dilution of serum that provided at serotype 10, and single calves were inoculated with serotypes least 50% protection of BHK-21 cell monolayers against 250 11, 13, and 17. Two additional calves were inoculated with TCID 50 of virus. The AGID test was performed according uninfected lysate of BHK-21 cells. Three lambs seronegative to standard protocol,i and results were reported as either to BTV, as determined by serum neutralization (SN) test and positive or negative. immune precipitation assay, 14 were raised in insect-secure isolation facilities and intravenously inoculated with 5 x 10 4 Results TCID 50 of BTV serotype 10. An additional lamb was inocThe epitope recognized by the MAb used in the ulated with a lysate of uninfected BHK-21 cells. Calves were cELISA was confirmed by direct immunofluorescence 1-20 wk old at inoculation, and lambs were approximately staining of virus-infected Vero cells to be present on 10 wk old. all 5 serotypes of BTV found in the United States but A panel of 30 bovine test sera was obtained and evaluated with the cELISA. Included in this panel were sera positive not on serotype 1 or 2 of EHDV. Hyperimmune rabbit and negative by BTV-AGID test and sera that contained antisera to each of the 5 serotypes of BTV inhibited neutralizing antibodies to EHDV serotypes 1 or 2 but not to binding (20%) of the MAb in the cELISA at final diBTV. An additional serum sample from a bull was also ob- lutions that ranged from 1:32, with antisera to serotype tained;f this serum gave a positive result with the BTV-AGID 13, to greater than 1: 1,024 with the antisera to serotest but contained only neutralizing antibody to EHDV and types 10, 11, and 17 (Table 1). The control rabbit not to BTV. antisera did not inhibit binding of the MAb at a diCompetitive ELISA test. The procedure used for the lution of 1:2. cELISA was a modification of a previously described methRuminant sera were used to compare the sensitivity od.2 ELISA plates were coated with a lysate of BTV serotype and specificity of the cELISA with those of the SN and lo-infected BHK-21 cells. At 36 hr after infection, the inAGID tests (Table 2). False positive results were not fected cells were subjected to a freeze/thaw cycle and diluted in 0.1 M NaHCO3 buffer. This antigen preparation was then obtained with any of the 3 serologic tests using sera used to coat round-bottom microtiter plates.g Test sera were from the control calves and lamb. Results obtained diluted in dilution buffer and added to antigen-coated wells with antisera from lambs inoculated with BTV seroprior to the addition of the biotinylated BTV-specific MAb. type 10 indicate that all 3 tests specifically identified After incubation for 1 hr, wells were washed and horseradish infected animals by 2 weeks after inoculation; howperoxidase-conjugated streptavidin was added and incubated ever, the cELISA appeared to be more sensitive than for 30 min. 2,2' Azino-bis(3-ethylbenzthiazoline-6-sulfonic either the AGID or SN tests. Serum obtained from acid (ABTS) substrate was added at a concentration of 0.22 lamb 3 at 7 days after inoculation, for instance, conmg/ml, and the reaction was stopped with sodium dodecyl sulfate (SDS) after approximately 10 min of incubation. End- tained neither precipitating nor neutralizing antibodies point optical density (OD) readings were made on a plate but gave a high degree of inhibition (77%) as deterreader h at 410 nm. Results obtained with the cELISA are mined by cELISA. Serum obtained from lamb 2 at 1 expressed as percent inhibition, as determined by the follow- week after inoculation was negative by the AGID test, whereas the same sample had a relatively high cELISA ing formula:
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146
Reddington, Reddington, MacLachlan Table 2. Comparison of serologic tests for bluetongue virus (BTV)-specific antibodies in sera from inoculated calves and lambs.
titer (39% inhibition). All 3 serologic tests also specif- BTV. An additional bovine serum that was BTV-AGID ically identified BTV-specific antibodies in sera from test positive, BTV-SN test negative, and EHDV-SN calves inoculated with each of the 4 different serotypes test positive also was determined to be cELISA test of BTV; all 5 infected calves were seropositive by the negative. These data strongly suggest that the AGID 3 tests at 2-6 weeks after inoculation. Sensitivity of test results from these 3 bovine sera are false positive the 3 tests varied among calves and with the serotype reactions and that the results of the cELISA and SN of BTV used. For instance, serum taken from calf 1 at tests are more valid. 4 weeks after inoculation had a high cELISA titer (58% inhibition) and a high SN titer (64) but was negative Discussion by the AGID test. Furthermore, serum taken from this Although the AGID test has served as the standard calf at 6 weeks after inoculation gave only a weak positive reaction with the AGID test, whereas the cELISA regulatory test for serologic diagnosis of BTV infection titer was high (78% inhibition). In contrast, serum taken in the United States and much of the rest of the world, from calf 4 (which was inoculated with BTV serotype it is increasingly obvious that the test is neither suffi13) at 4 weeks after inoculation was positive by the ciently sensitive nor specific to justify its continued 2,3,6 AGID test and had a high SN titer (64) but had a low use Our results confirm that the cELISA test is both more sensitive and more specific than the AGID test, (19% inhibition) titer as determined by cELISA. The cELISA was further evaluated using a panel of which is to be expected because the cELISA test quancattle sera obtained from the National Animal Disease titates only the displacement of BTV-specific MAb by Center. The cELISA confirmed results of the AGID the test serum. Problems with epitopes common to 10 test with 2 exceptions; both these sera were positive •BTV and closely related serogroups that produce false by the AGID test, negative by cELISA, and both sera positive reactions with the AGID test are thus avoided. contained neutralizing antibodies to EHDV but not to In addition, the cELISA is quantitative and avoids
Downloaded from vdi.sagepub.com at NEW YORK UNIV LIBRARY on April 16, 2015
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subjective interpretation, which is inherent in the interpretation of the AGID test. Results of this study indicate that sensitivity of the cELISA is comparable to that of the SN test; the SN test is generally accepted as the most sensitive and specific of the available tests for serologic detection of BTV infection of ruminants. The SN test is too cumbersome and laborious for routine use in the diagnostic laboratory, especially when sera are to be screened for the presence of antibodies to multiple serotypes of BTV. Comparison of the results of this study with data obtained previously with some of the same sera also indicate that the as presented here has comparable sensitivity to that of the Western immunoblot and immune precipitation assays.14 Immunoblotting and especially immune precipitation are highly sensitive and specific methods of assaying for the presence of viral-specific polypeptides, but, like the SN test, they are not appropriate for routine diagnostic use. Any used for serologic diagnosis of BTV in the United States obviously must incorporate an MAb directed at an epitope present on all serotypes of BTV found in the United States. In addition, the epitope recognized by the MAb must be unique to BTV serogroup viruses. Results of this and prior studies clearly indicate that the VP7-specific 290 is well suited for use in the VP7 is a component of the core of the BTV virion and is considered a group-specific protein, 7 which is compatible with our observation that the epitope recognized by our VP7-specific is present on all serotypes of BTV found in the United States. Bluetongue virus-infected cattle and sheep mount a strong humoral immune response to VP7;14 therefore, a that detects antibodies specific for VP7 is a logical test for serologic confirmation of BTV infection of ruminants. In summary, the is a superior test for serologic diagnosis of BTV infection of ruminants in the United States because it has comparable sensitivity to the SN test, requires significantly less time to run, and provides objective results. Sources and manufacturers a. BluePlate Special, Consultants for Applied Biosciences, Wilton, CT. b. Supplied by Dr. J. L. Stott, School of Veterinary Medicine, University of California, Davis, CA. c. Pierce, Rockford, IL. d. Zymed Laboratories, South San Francisco, CA. e. United States Department of Agriculture, National Veterinary Services Laboratory, Ames, IA.
f. Supplied by Dr. B. I. Osbum, School of Veterinary Medicine, University of California, Davis, CA. g. Falcon Microtest III, Becton Dickinson and Co., Lincoln Park, NJ. h. MR 5000, Dynatech Laboratories, Chantilly, VA. i. California Veterinary Diagnostic Laboratory, Davis, CA.
References 1. 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. 2. Ashfar A, Thomas FC, Wright PF, et al.: 1987, Comparison of competitive and indirect enzyme-linked immunosorbent assays for detection of bluetongue virus antibodies in serum and whole blood. J Clin Microbiol 25: 1705-1710. 3. Ashfar A, Thomas FC, Wright PF, et al.: 1989, Comparison of competitive ELISA, indirect ELISA, and standard AGID tests for detecting bluetongue virus antibodies in cattle and sheep. Vet Ret 124:136-141. 4. Borden EC, Shope RE, Murphy FA: 1971, Physiochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus-a new taxonomic group: physiochemical and serological studies. J Gen Virol 13:262-271. 5. Bowne JG: 1971, Bluetongue disease. Adv Vet Sci Comp Med 15: 1-46. 6. Della-Porta AJ, Parsonson IM, McPhee DA: 1985, Problems in the interpretation of diagnostic tests due to cross-reactions between orbiviruses and broad serological responses in animals. Prog Clin Biol Res 178:445-454. 7. French TJ, Roy P: 1990, Synthesis of bluetongue virus (BTV) core-like particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. J Virol 64: 15301536. 8. Heidner HW, MacLachlan NJ, Fuller FJ, et al.: 1988, Bluetongue virus genome remains stable during prolonged infection of cattle. J Gen Virol 69:2629-2636. 9. House C, House JA, Beminger ML: 1990, Detection of bluetongue group-specific antibody by competitive ELISA. J Vet Diagn Invest 2: 137-139. 10. Huismans H, Bremer CW, Barber TL: 1979, The nucleic acid and proteins of epizootic haemorrhagic disease virus. Onderstepoort J Vet Res 46:95-104. 11. Lunt RA, White JR, Blacksell SD: 1988, Evaluation of a 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. 12. MacLachlan NJ, Schore CE, Osbum BI: 1984, Antiviral responses of bluetongue virus inoculated bovine fetuses and their dams. Am J Vet Res 45:1469-1473. 13. Osbum BI: 1988, Economics of bluetongue in the United States. Proc 4th Symp Arbovirus Res Aust, pp. 245-247. 14. Richards RG, MacLachlan NJ, Heidner HW, et al.: 1988, Comparison of virologic and serologic responses of lambs and calves infected with bluetongue virus serotype 10. Vet Microbiol 18: 233-242. 15. Whetter LE, MacLachlan NJ, Gebhard DH, et al.: 1989, Bluetongue virus infection of bovine monocytes. J Gen Virol 70: 1663-1676.
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