Sensitivity and Specificity of an Enzyme-linked Immunosorbent Assay for the Detection of Bovine Viral Diarrhea Virus Antibody in Cattle Hyun J. Cho, Saad A. Masri, Dirk Deregt, Sang-Geon Yeo and Elizabeth J. Golsteyn Thomas
ABSTRACT
RESUME
INTRODUCTION
A reliable bovine viral diarrhea (BVD) viral antigen was prepared from BVD virus grown on Madin Darby bovine kidney (MDBK) cells by solubilizing the virus with detergent MEGA-10 (decanoyl-N-methylglucamide) followed by removal of hydrophobic proteins with Triton X-100 treatment. By these treatments, problems of high background associated with BVD viral antigen in the enzyme-linked immunosorbent assay (ELISA) were eliminated. With this new antigen, an ELISA was adapted to detect bovine serum antibody against BVD virus. The diagnostic specificity of the assay in 403 bovine sera collected from a BVD virus-free herd was 100%; in 296 bovine sera with serum neutralizing antibody titers of > 1:2, 289 sera were ELISA positive (relative sensitivity of 97.6%), two sera gave false negative reactions (0.7%) and five sera gave suspicious reactions (1.7%). These interpretations were based on positive/negative (P/N) ratio readings, i.e. a P/N ratio of < 1.50, 1.50-1.99 and > 2.00 were interpreted as negative, suspicious and positive reactions, respectively. The ELISA results gave excellent agreement with serum neutralization in detecting both seropositive and seronegative animals (Kappa = 0.994). The ELISA assay was considered to be technically superior to the serum neutralization test for the routine detection of BVD viral antibody in bovine sera.
Un antigene du virus de la diarrhee a virus du bovin (BVD) a ete obtenu a partir d'un milieu de culture Madin Darby de cellules renales bovines. L'antigene a par la suite ete solubilise avec un detergent, le MEGA-10, et extrait des proteines hydrophobiques avec du Triton X-100. Ces traitements ont permis une meilleure efficacite du test ELISA a cause dune reduction du bruit de fond lors de la lecture. Suite a l'obtention de ce nouvel antigene, un test diagnostique pour la detection d'anticorps seriques anti-BVD a ete mis au point. La specificite du test a ete de 100% lorsque mesuree sur 403 echantillons de serum provenant d'un troupeau exempt de BVD. Des 296 echantillons seriques ayant un titre d'anticorps neutralisants > 1:2, 289 echantillons se sont averes positifs (sensitivite de 97.6%), deux echantillons ont donne des resultats faussement netgatifs (0.7%) et cinq echantillons ont donne des resultats douteux (1.7%). Ces resultats sont bases sur le ratio de lectures positives/negatives (P/N) i.e. un ratio P/N > 1.50, de 1.50 a 1.99 et > 2.00 ont ete interpretes comme etant respectivement negatif, douteux et positif. Les resultats de ce test ELISA ont permis de detecter autant les animaux seropositifs que seronegatifs (Kappa = 0.994). De plus, ce test peut etre considere comme etant superieur au test de seroneutralisation conventionnel utilise pour la detection d'anticorps seriques anti-
The serum neutralization (SN) and immunoperoxidase (IP) tests are routinely used by Agriculture Canada
laboratories for the detection of bovine viral diarrhea (BVD) viral antibody and BVD virus, respectively. The SN test is an effective test with high specificity, but has certain limitations. It requires expensive tissue culture systems and is time consuming. Therefore, an alternative specific, sensitive and reliable in vitro method is desirable for the detection of BVD viral antibody in cattle sera and for export certification purposes. During the last several years, several reports on the enzyme-linked immunosorbent assay (ELISA) for the detection of BVD viral antibody have been published (1-5). Some of these tests have proved to be unreliable for routine diagnostic purposes, and have presented difficulties in the attachment of viral antigen to microtiter plates or problems with a high background reading when tested with polyclonal secondary antibody as a conjugate. We report here the production of a reliable BVD viral antigen which is readily attached to solid phase without these high background problems. This communication describes the application of an ELISA for detection -of BVD viral antibodies in bovine sera using the new BVD viral antigen, and measures the performance of the test in terms of its
BVD. (Traduit par Dr. Pascal Dubreuil)
diagnostic specificity and sensitivity.
Agriculture Canada, Animal Diseases Research Institute, P.O. Box 640, Lethbridge, Alberta TIJ 3Z4. Dr. Sang-Geon Yeo is a visiting scientist from Kyungsang National University, South Korea and Dr. E.J. Golsteyn Thomas is a Canadian Government Laboratory Visiting Fellow. Submitted April 30, 1990.
56
Can J Vet Res 1991; 55: 56-59
MATERIALS AND METHODS SERA
Four hundred and three sera collected from the Animal Diseases Research Institute (ADRI) Lethbridge cattle herd were used to determine the diagnostic specificity of the ELISA. All cattle in the herd have been BVD viral SN antibody negative for three consecutive biannual tests. For the sensitivity assay of the ELISA, 296 bovine sera with SN titers > 1:2 were obtained from a serum bank kept at this laboratory. SERUM NEUTRALIZATION TEST
Standard SN tests were performed using the NADL strain of BVD virus in microtiter plates (6). Twofold serial dilutions (1:2-1:256 or 1:5-1:320) of heat-inactivated serum (560 C, 30 min) were mixed with equal volumes (0.05 mL) of BVD virus, containing 100 median tissue culture infectious doses (TCID50). The mixtures were incubated for 1 h at 370 C in a CO2 incubator. At the end of incubation, 0.05 mL of secondary bovine (calf) kidney cells in Eagle's minimal essential medium (MEM) (Gibco Canada Inc., Burlington, Ontario) supplemented with 20% calf serum were added to the test wells and the microplates were further incubated for five days. The highest dilution of serum which gave complete neutralization of the virus was recorded as the SN titer. PROPAGATION OF BVD VIRUS AND PRODUCTION OF BVD VIRAL ANTIGEN
370C for 2 h on a rocking platform (Bellco, Vinelane, New Jersey). The virus inoculum was removed and replaced with 20 mL of MEM without serum. Three days after infection, when virus-induced cytopathic effects had reached a maximum, the infected cells and culture supernatant were collected and centrifuged at 1600 x g for 30 min. The resulting cell pellets were separated from the supernatant. The cell pellets were resuspended to 1 / 200 of their original volume in 0.85% saline. The supernatant received a final concentration of 8% polyethylene glycol (molecular weight 70009000) (Sigma, St. Louis, Missouri) and 0.5 M NaCl and was stirred overnight at 40 C followed by centrifugation at 3300 x g for 30 min. The supernatant was discarded and the pellet was dissolved as described above. Suspensions from both pellets were combined and MEGA-10 (Calbiochem) detergent (decanoyl-Nmethylglucamide) at final concentration of 1% was added as described (5) and sonicated until the detergent completely solubilized. The lysate was vigorously shaken at 370C for 1 h and then Triton X-100 (final concentration 0. 1%) was added and shaken for another hour at 370 C. The treated BVD viral antigen was centrifuged at 3000 x g for 30 min, the supernatant was collected, 500 mg of Bio-beads SM-2 adsorbent (Bio-Rad) were added and the suspension was 7-
dialyzed against large volumes of saline overnight at 40 C. The BVD viral antigen and the Bio-beads SM-2 mixtures were further stirred for 1 h at 40 C and centrifuged at 3000 x g for 30 min. The supernatant obtained was used as the source of BVD viral antigen for use in the ELISA. ELISA PROCEDURES
Linbro enzyme immunoassay microtitration plates with 96 flat bottom wells (Flow Laboratories, Hamden, Connecticut, Catalogue No. 76-381-04) were used for the test. The working dilution of the antigen was made in 0.05 M carbonate buffer with 0.125 M NaCl at pH 9.5. The wells of the microtitration plates were sensitized by adding 0.2 mL of the diluted antigen per well. After incubation at 40 C overnight in a covered container, the wells were decanted and blocked by adding 0.35 mL diluent [Dulbecco phosphate buffered saline (PBS), containing 0.5% Tween 20 and 1% heat inactivated (560 C for 30 min) horse serum] to each well. After incubation for 10-15 min at room temperature, the plates were washed three times with deionized water in a Titertek microplate washer (Flow Laboratories, Hamden, Connecticut). Sera were diluted 1:100 in the same diluent and tested in volumes of 200 ,uL per well in duplicate. Sample placement configuration is illustrated in Fig. 1. Following an incubation period of 2 h
-r
-r
0 0 0 00 0 (2)80000 0 0 00 0 2~6~2)QQQQ6) 000 0 0 0 0 0 0 000 0 000 80000 © 0 0 0 000 0
The Singer strain of BVD virus was plaque-purified and then further purified by the limiting-dilution (23)QQQ method. Specifically, stock virus was (obtained from microtiter wells containing a single focus of virus-infected cells as determined by an indirect 7- ~~~~'r i immunoperoxidase test (7). Bovine viral diarrhea virus-free Madin-Darby bovine kidney (MD BK) cells were grown in 150 cm2 flasks (Corning) to confluency. Minimal essential medium supplemented with 5% horse serum (Gibco) was used as the growth medium. At cell confluency, the medium was removed and the cell monolayer was incubated with Fig. 1. Sample placement configuration in 96 well plate. A: Reference positive serum (single serum 0.03 mL of virus (approximate titer of with SN titer of 1:20,000). B,C: Reference negative serum (a pool of 30 SN negative sera). D: Diluent 107 TCID50/mL) in 5 mL of MEM at buffer control. 1-40: Test sample duplicates, 1:100 dilution. -
57
at 370 C, the plates were washed five times as described above. Peroxidase conjugated IgG fraction of rabbit antibovine IgG (heavy and light chains) was diluted in the diluent (1:20,000 dilution with Lot No. 22112 conjugate, Cappel Laboratories, Cochranville, Pennsylvania) and 0.2 mL of the conjugate was dispensed into each well and allowed to react for 1 h at 370 C, followed by washing three times in the microplate washer. Freshly prepared 0-phenylenediamine dihydrochloride (Sigma), 1 mg/ mL in 0.1 M citrate buffer pH 5.5 with 3 mM H202 was used as the substrate. After the substrate (0.2 mL per well) had been incubated for 45 min at room temperature, 50 ,uL of 4 N H2SO4 were added to each well to stop the reaction. The absorbance at 492 nm (A492) value of each plate was read with a Titertek Multiskan photometer (Flow Laboratories). The serological status of each test sample was determined from the P/N ratio (positive/ negative ratio) which is the mean optical density (OD) of the duplicate sample divided by the mean OD of eight replicates of the reference negative serum (a pool of 30 negative samples as determined by SN). The test sera were assigned a status of positive (P/N ratio > 2.00), suspicious (P/N ratio > 1.50 < 2.00) or negative (P/N ratio < 1.50). The performance of the ELISA was considered in terms of the following parameters as defined by Vecchio (8): Specificity (%) = No. of noninfected cattle with a negative test x 100 all noninfected cattle tested Sensitivity (%) = No. of infected cattle with a positive test x 100 all infected cattle tested
2.5
FREQUENCY OF DISTRIBUTION OF A492 VALUE AND P/N RATIOS OF NORMAL BOVINE SERA AND SPECIFICITY OF ELISA
2.0
1.5 C\J 0)
1.0*
0.5 -.-
020
40
.
-0- -I---
-,
80 160 320 640 1280
Antigen dilution Fig. 2. Bovine virus diarrhea viral ELISA antigen titration with positive (solid line) and negative (broken line) reference sera. A492 absorbancy at 492 nm.
progressively higher A492 values. With a negative reference serum the antigen dilutions did not affect the A492 value significantly. When BVD viral antigens were prepared according to a previously reported protocol (5) (i.e. solubilization of the BVD virus pellet with PBS containing 1% of the detergent MEGA-10, sonication and fractionation on Sephadex G-50 column), high background readings with negative reference sera were observed when tested with polyclonal second antibody conjugate. These background readings (A492 values) ranged between 0.250 to 0.350. These background readings were too high since the minimum cut-off value for positive reactions (twice the negative value) would have a range of 0.500 to 0.700 (A492 value) which would give a high proportion of positive sera as STATISTICS false negative. With the present Agreement between ELISA and SN method of antigen preparation, these tests was measured using Kappa high background reactions with Statistics (9) using 699 sera (403 SN negative sera were eliminated and negative and 296 SN positive sera). furthermore the BVD viral antigen was readily attached to the microtitration plates. RESULTS Negative control antigen prepared from the mock infected MDBK cells ELISA ANTIGEN Figure 2 shows a typical titration as described for the BVD viral antigen curve of the BVD viral ELISA anti- did not show significant reactivity gen. With a positive reference serum, with both positive and negative higher concentrations of antigen gave reference sera (data not shown). 58
Table I illustrates the A492 value and P/N ratio frequency distribution of 403 bovine sera against the BVD virus ELISA antigen. The sera were collected from a BVD virus-free herd at ADRI. The mean A492 value was 0.138 + 0.030 and the mean P/N ratio was 1.016 ± 0.102. If a P/N ratio of less than 1.50 was used as the cut-off value for negative, the specificity of this ELISA in 403 BVD virus-free bovine sera was 100%. RELATIVE SENSITIVITY OF ELISA
Two hundred and ninety-six sera which had BVD virus SN antibody titers of > 1:2 were used to determine the relative sensitivity of the present ELISA (Table II). Of these sera, 289 sera were ELISA positive and two sera were ELISA negative (false negative 0.7%), and there were five sera (1.7%) with suspicious reactions. Thus, the relative sensitivity of the present ELISA was 97.6%. KAPPA STATISTICS
Six hundred and ninety-nine sera were used to determine the agreement between ELISA and SN tests. Table III illustrates the Kappa statistics. The TABLE I. Frequency distribution of A492 value and P/N ratio of normal bovine sera (n = 403) against BVD viral (Singer Strain) antigen in the ELISA Absorbance at 492 nm (A492) < 0.074 0.075 - 0.099 0.100-0.124 0.125 -0.149 0.150 - 0.174 0.175-0.199 0.200 - 0.224 > 0.225 Total Mean ± SD = 0.1380 ± 0.30
No. of sera 6 54 54 140 106 38 5 0 403
P/N Ratio 0.70 - 0.89 0.90- 1.09 1.10-1.29 1.30- 1.49 >1.50 Total
No. of sera 41 274 86 2 0 403
Mean±5D= 1.016±0.102
TABLE II. Comparison of ELISA with SN test
ELISAb results Nega- Suspi- Positive cious tive No. SNa titer 24 2 1:2 - 1:10 29 3 2 1:16- 1:40 83 0 81 0 59 1:64- 1:80 59 0 > 1:128 0 125 125 0 2 Total 5 289 296 aSerum neutralization test for BVD virus antibodies bEnzyme-linked immunosorbent assay
ELISA results gave an excellent agreement with SN in detecting both seropositive and seronegative animals. The Kappa value was 0.994 with a 95% confidence limit of 0. 92-1.00.
DISCUSSION In the present study, BVD viral antigen for use in the ELISA was prepared first by solubilizing BVD virus infected cell pellets with the detergent MEGA-10 as described by Juntti et al (5). The inherent problem of high background reactions of the antigen in ELISA was eliminated by further treatment of the solubilized antigen with Triton X-100 and by removing the Triton X-100 bound proteins and free Triton X-100 by centrifugation and adsorption with Bio-beads SM-2. The Triton X series of nonionic detergents has been known to bind preferentially to hydrophobic proteins (10,11). Thus, hydrophobic proteins (bound to Triton X-100), which were the probable cause of nonspecific high background reactions were eliminated or remained masked, and predominantly hydrophilic proteins were enriched in the present BVD viral antigen. Nonionic detergents such as Triton X-100 and Nonidet P40 (NP40) have been shown to retard or prevent protein binding to microtiter wells (1214). Therefore, their removal during antigen preparation is desired. It has been shown previously that there was considerable difficulty with NP40 treated BVD viral antigen in the attachment of the antigen on ELISA plates (2,3). In another study, when NP40 solubilized porcine epidemic diarrhea coronavirus was used for an ELISA, no reactions were detected at low dilutions of antigen and only when antigen dilutions greater than 1:150
TABLE III. Comparison of ELISA and SN tests (n = 699) by a 2 x 2 table and Kappa statistics
SNa Category +
ELISAb
_
+
-
294c 2
0 403
Sum 294 405
699 296 403 Sum = 0.997 Agreement observed = 0.512 Agreement due to chance = 0.994 Kappa = 0.038 SE (K) = 0.92 - 1.00 95% Confidence limits (K) = 26.28 Kappa/ SE(K) aSerum neutralization test for BVD virus
antibodies bEnzyme-linked immunosorbent assay cELISA suspicious reactions (N = 5) were considered as ELISA positive category in calculating the Kappa statistics
were used was an optical density of 0.9 reached (14). As seen in Fig. 2, the present BVD viral antigen did not cause high background reaction problems and readily attached to the microtiter plates and thus enabled us to detect BVD viral antibody in bovine sera. Based on the results obtained, the ELISA method described here appears to be an efficient serological test in terms of excellent specificity and sensitivity. It agrees closely with the SN test in detecting both seropositive and seronegative animals (Kappa = 0.994). The test is also more practical and faster than the SN test for the detection of BVD viral antibody. Because of its many advantages over the SN test, such as its convenient technical features, together with its
potential adaptability for automation, this ELISA method may have value as an alternative test for the detection of BVD viral antibodies in cattle. ACKNOWLEDGMENTS The authors thank Mr. P. Mohanraj for his excellent assistance with the ELISA and Mrs. S.E. Smithson and Mr. W.A. Pickering for assistance with cell culture and serum neutralization tests. Drs. V.W. Lees and K.G. Loewen supplied many of the serum samples used in this study and reference positive sera. Dr. V.W. Lees also gave valuable advice on Kappa statistics. We are also grateful to Ms. S.M. Braun for preparing the figures and Miss S. Van Dyk for assistance in the manuscript preparation.
REFERENCES 1. CHU HJ, ZEE YC, ARDANS AA, DAI K. Enzyme-linked immunosorbent assay for detection of antibodies to bovine viral diarrhea virus in bovine sera. Vet Microbiol
1985; 10: 325-333. 2. HOWARD CJ, CLARKE MC, BROWNLIE J. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle sera. Vet Microbiol 1985; 10: 359-369. 3. BOCK RE, BURGESS GW, DOUGLAS IC. Development of an enzyme-linked immunosorbent assay (ELISA) for the detection of bovine serum antibody to bovine viral diarrhoea virus. Aust Vet J
1986; 63: 406-408. 4. JUSTEWICZ DM, MAGAR R, MARSOLAIS G, LECOMTE J. Bovine viral diarrhea virus-infected MDBK monolayer as antigen in enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies in bovine sera. Vet Immunol Immunopathol 1987; 14: 377-384. 5. JUNTTI N, LARSSON B, FOSSUM C. The use of monoclonal antibodies in enzyme linked immunosorbent assays for detection of antibodies to bovine viral diarrhoea virus. J Vet Med B 1987; 34: 356-363. 6. CARBREY EA. Recommended standard laboratory techniques for diagnosing infectious bovine rhinotracheitis, bovine virus diarrhea and shipping fever (parainfluenza 3). Proc US Anim Health Assoc 1971; 75: 629-648. 7. DEREGT D, MASRI SA, CHO HJ, BIELEFELDT OHMANN H. Monoclonal antibodies to the p80/ 125 and gp53 proteins of bovine viral diarrhea virus: Their potential use as diagnostic reagents. Can J Vet Res 1990; 54: 343-348. 8. VECCHIO TJ. Predictive value of a single diagnostic test in unselected populations. New Engl J Med 1966; 274: 1171-1173. 9. MARTIN SW, MEEK AH, WILLEBERG P. Veterinary Epidemiology: Principles and Methods. Ames: Iowa State University Press, 1987: 74. 10. BORDIER C. Phase separation of integral membrane proteins in Triton X-1 14 solution. J Biol Chem 1981; 256: 1604-1607. 11. PRYDE J. Triton X-1 14: a detergent that has come in from the cold. Trends Biochem Sci 1986; 11: 160-163. 12. PALFREE RG, ELLIOTT BE. An enzyme-linked immunosorbent assay (ELISA) for detergent solubilized Ia glycoproteins using nitrocellulose membrane disks. J Immunol Methods 1982; 52: 395-408. 13. DREKLER G, EICHINGER A, WOLF C, SIEGHARD W. A rapid and simple method for efficient coating of microtiter plates using low amounts of antigen in the presence of detergent. J Immunol Methods 1986; 95: 117-122. R. WYLER M, 14. HOFMANN Enzyme-linked immunosorbent assay for the detection of porcine epidemic diarrhea coronavirus antibodies in swine sera. Vet Microbiol 1990; 21: 263-273.
59
ABSTRACT
RESUME
INTRODUCTION
A reliable bovine viral diarrhea (BVD) viral antigen was prepared from BVD virus grown on Madin Darby bovine kidney (MDBK) cells by solubilizing the virus with detergent MEGA-10 (decanoyl-N-methylglucamide) followed by removal of hydrophobic proteins with Triton X-100 treatment. By these treatments, problems of high background associated with BVD viral antigen in the enzyme-linked immunosorbent assay (ELISA) were eliminated. With this new antigen, an ELISA was adapted to detect bovine serum antibody against BVD virus. The diagnostic specificity of the assay in 403 bovine sera collected from a BVD virus-free herd was 100%; in 296 bovine sera with serum neutralizing antibody titers of > 1:2, 289 sera were ELISA positive (relative sensitivity of 97.6%), two sera gave false negative reactions (0.7%) and five sera gave suspicious reactions (1.7%). These interpretations were based on positive/negative (P/N) ratio readings, i.e. a P/N ratio of < 1.50, 1.50-1.99 and > 2.00 were interpreted as negative, suspicious and positive reactions, respectively. The ELISA results gave excellent agreement with serum neutralization in detecting both seropositive and seronegative animals (Kappa = 0.994). The ELISA assay was considered to be technically superior to the serum neutralization test for the routine detection of BVD viral antibody in bovine sera.
Un antigene du virus de la diarrhee a virus du bovin (BVD) a ete obtenu a partir d'un milieu de culture Madin Darby de cellules renales bovines. L'antigene a par la suite ete solubilise avec un detergent, le MEGA-10, et extrait des proteines hydrophobiques avec du Triton X-100. Ces traitements ont permis une meilleure efficacite du test ELISA a cause dune reduction du bruit de fond lors de la lecture. Suite a l'obtention de ce nouvel antigene, un test diagnostique pour la detection d'anticorps seriques anti-BVD a ete mis au point. La specificite du test a ete de 100% lorsque mesuree sur 403 echantillons de serum provenant d'un troupeau exempt de BVD. Des 296 echantillons seriques ayant un titre d'anticorps neutralisants > 1:2, 289 echantillons se sont averes positifs (sensitivite de 97.6%), deux echantillons ont donne des resultats faussement netgatifs (0.7%) et cinq echantillons ont donne des resultats douteux (1.7%). Ces resultats sont bases sur le ratio de lectures positives/negatives (P/N) i.e. un ratio P/N > 1.50, de 1.50 a 1.99 et > 2.00 ont ete interpretes comme etant respectivement negatif, douteux et positif. Les resultats de ce test ELISA ont permis de detecter autant les animaux seropositifs que seronegatifs (Kappa = 0.994). De plus, ce test peut etre considere comme etant superieur au test de seroneutralisation conventionnel utilise pour la detection d'anticorps seriques anti-
The serum neutralization (SN) and immunoperoxidase (IP) tests are routinely used by Agriculture Canada
laboratories for the detection of bovine viral diarrhea (BVD) viral antibody and BVD virus, respectively. The SN test is an effective test with high specificity, but has certain limitations. It requires expensive tissue culture systems and is time consuming. Therefore, an alternative specific, sensitive and reliable in vitro method is desirable for the detection of BVD viral antibody in cattle sera and for export certification purposes. During the last several years, several reports on the enzyme-linked immunosorbent assay (ELISA) for the detection of BVD viral antibody have been published (1-5). Some of these tests have proved to be unreliable for routine diagnostic purposes, and have presented difficulties in the attachment of viral antigen to microtiter plates or problems with a high background reading when tested with polyclonal secondary antibody as a conjugate. We report here the production of a reliable BVD viral antigen which is readily attached to solid phase without these high background problems. This communication describes the application of an ELISA for detection -of BVD viral antibodies in bovine sera using the new BVD viral antigen, and measures the performance of the test in terms of its
BVD. (Traduit par Dr. Pascal Dubreuil)
diagnostic specificity and sensitivity.
Agriculture Canada, Animal Diseases Research Institute, P.O. Box 640, Lethbridge, Alberta TIJ 3Z4. Dr. Sang-Geon Yeo is a visiting scientist from Kyungsang National University, South Korea and Dr. E.J. Golsteyn Thomas is a Canadian Government Laboratory Visiting Fellow. Submitted April 30, 1990.
56
Can J Vet Res 1991; 55: 56-59
MATERIALS AND METHODS SERA
Four hundred and three sera collected from the Animal Diseases Research Institute (ADRI) Lethbridge cattle herd were used to determine the diagnostic specificity of the ELISA. All cattle in the herd have been BVD viral SN antibody negative for three consecutive biannual tests. For the sensitivity assay of the ELISA, 296 bovine sera with SN titers > 1:2 were obtained from a serum bank kept at this laboratory. SERUM NEUTRALIZATION TEST
Standard SN tests were performed using the NADL strain of BVD virus in microtiter plates (6). Twofold serial dilutions (1:2-1:256 or 1:5-1:320) of heat-inactivated serum (560 C, 30 min) were mixed with equal volumes (0.05 mL) of BVD virus, containing 100 median tissue culture infectious doses (TCID50). The mixtures were incubated for 1 h at 370 C in a CO2 incubator. At the end of incubation, 0.05 mL of secondary bovine (calf) kidney cells in Eagle's minimal essential medium (MEM) (Gibco Canada Inc., Burlington, Ontario) supplemented with 20% calf serum were added to the test wells and the microplates were further incubated for five days. The highest dilution of serum which gave complete neutralization of the virus was recorded as the SN titer. PROPAGATION OF BVD VIRUS AND PRODUCTION OF BVD VIRAL ANTIGEN
370C for 2 h on a rocking platform (Bellco, Vinelane, New Jersey). The virus inoculum was removed and replaced with 20 mL of MEM without serum. Three days after infection, when virus-induced cytopathic effects had reached a maximum, the infected cells and culture supernatant were collected and centrifuged at 1600 x g for 30 min. The resulting cell pellets were separated from the supernatant. The cell pellets were resuspended to 1 / 200 of their original volume in 0.85% saline. The supernatant received a final concentration of 8% polyethylene glycol (molecular weight 70009000) (Sigma, St. Louis, Missouri) and 0.5 M NaCl and was stirred overnight at 40 C followed by centrifugation at 3300 x g for 30 min. The supernatant was discarded and the pellet was dissolved as described above. Suspensions from both pellets were combined and MEGA-10 (Calbiochem) detergent (decanoyl-Nmethylglucamide) at final concentration of 1% was added as described (5) and sonicated until the detergent completely solubilized. The lysate was vigorously shaken at 370C for 1 h and then Triton X-100 (final concentration 0. 1%) was added and shaken for another hour at 370 C. The treated BVD viral antigen was centrifuged at 3000 x g for 30 min, the supernatant was collected, 500 mg of Bio-beads SM-2 adsorbent (Bio-Rad) were added and the suspension was 7-
dialyzed against large volumes of saline overnight at 40 C. The BVD viral antigen and the Bio-beads SM-2 mixtures were further stirred for 1 h at 40 C and centrifuged at 3000 x g for 30 min. The supernatant obtained was used as the source of BVD viral antigen for use in the ELISA. ELISA PROCEDURES
Linbro enzyme immunoassay microtitration plates with 96 flat bottom wells (Flow Laboratories, Hamden, Connecticut, Catalogue No. 76-381-04) were used for the test. The working dilution of the antigen was made in 0.05 M carbonate buffer with 0.125 M NaCl at pH 9.5. The wells of the microtitration plates were sensitized by adding 0.2 mL of the diluted antigen per well. After incubation at 40 C overnight in a covered container, the wells were decanted and blocked by adding 0.35 mL diluent [Dulbecco phosphate buffered saline (PBS), containing 0.5% Tween 20 and 1% heat inactivated (560 C for 30 min) horse serum] to each well. After incubation for 10-15 min at room temperature, the plates were washed three times with deionized water in a Titertek microplate washer (Flow Laboratories, Hamden, Connecticut). Sera were diluted 1:100 in the same diluent and tested in volumes of 200 ,uL per well in duplicate. Sample placement configuration is illustrated in Fig. 1. Following an incubation period of 2 h
-r
-r
0 0 0 00 0 (2)80000 0 0 00 0 2~6~2)QQQQ6) 000 0 0 0 0 0 0 000 0 000 80000 © 0 0 0 000 0
The Singer strain of BVD virus was plaque-purified and then further purified by the limiting-dilution (23)QQQ method. Specifically, stock virus was (obtained from microtiter wells containing a single focus of virus-infected cells as determined by an indirect 7- ~~~~'r i immunoperoxidase test (7). Bovine viral diarrhea virus-free Madin-Darby bovine kidney (MD BK) cells were grown in 150 cm2 flasks (Corning) to confluency. Minimal essential medium supplemented with 5% horse serum (Gibco) was used as the growth medium. At cell confluency, the medium was removed and the cell monolayer was incubated with Fig. 1. Sample placement configuration in 96 well plate. A: Reference positive serum (single serum 0.03 mL of virus (approximate titer of with SN titer of 1:20,000). B,C: Reference negative serum (a pool of 30 SN negative sera). D: Diluent 107 TCID50/mL) in 5 mL of MEM at buffer control. 1-40: Test sample duplicates, 1:100 dilution. -
57
at 370 C, the plates were washed five times as described above. Peroxidase conjugated IgG fraction of rabbit antibovine IgG (heavy and light chains) was diluted in the diluent (1:20,000 dilution with Lot No. 22112 conjugate, Cappel Laboratories, Cochranville, Pennsylvania) and 0.2 mL of the conjugate was dispensed into each well and allowed to react for 1 h at 370 C, followed by washing three times in the microplate washer. Freshly prepared 0-phenylenediamine dihydrochloride (Sigma), 1 mg/ mL in 0.1 M citrate buffer pH 5.5 with 3 mM H202 was used as the substrate. After the substrate (0.2 mL per well) had been incubated for 45 min at room temperature, 50 ,uL of 4 N H2SO4 were added to each well to stop the reaction. The absorbance at 492 nm (A492) value of each plate was read with a Titertek Multiskan photometer (Flow Laboratories). The serological status of each test sample was determined from the P/N ratio (positive/ negative ratio) which is the mean optical density (OD) of the duplicate sample divided by the mean OD of eight replicates of the reference negative serum (a pool of 30 negative samples as determined by SN). The test sera were assigned a status of positive (P/N ratio > 2.00), suspicious (P/N ratio > 1.50 < 2.00) or negative (P/N ratio < 1.50). The performance of the ELISA was considered in terms of the following parameters as defined by Vecchio (8): Specificity (%) = No. of noninfected cattle with a negative test x 100 all noninfected cattle tested Sensitivity (%) = No. of infected cattle with a positive test x 100 all infected cattle tested
2.5
FREQUENCY OF DISTRIBUTION OF A492 VALUE AND P/N RATIOS OF NORMAL BOVINE SERA AND SPECIFICITY OF ELISA
2.0
1.5 C\J 0)
1.0*
0.5 -.-
020
40
.
-0- -I---
-,
80 160 320 640 1280
Antigen dilution Fig. 2. Bovine virus diarrhea viral ELISA antigen titration with positive (solid line) and negative (broken line) reference sera. A492 absorbancy at 492 nm.
progressively higher A492 values. With a negative reference serum the antigen dilutions did not affect the A492 value significantly. When BVD viral antigens were prepared according to a previously reported protocol (5) (i.e. solubilization of the BVD virus pellet with PBS containing 1% of the detergent MEGA-10, sonication and fractionation on Sephadex G-50 column), high background readings with negative reference sera were observed when tested with polyclonal second antibody conjugate. These background readings (A492 values) ranged between 0.250 to 0.350. These background readings were too high since the minimum cut-off value for positive reactions (twice the negative value) would have a range of 0.500 to 0.700 (A492 value) which would give a high proportion of positive sera as STATISTICS false negative. With the present Agreement between ELISA and SN method of antigen preparation, these tests was measured using Kappa high background reactions with Statistics (9) using 699 sera (403 SN negative sera were eliminated and negative and 296 SN positive sera). furthermore the BVD viral antigen was readily attached to the microtitration plates. RESULTS Negative control antigen prepared from the mock infected MDBK cells ELISA ANTIGEN Figure 2 shows a typical titration as described for the BVD viral antigen curve of the BVD viral ELISA anti- did not show significant reactivity gen. With a positive reference serum, with both positive and negative higher concentrations of antigen gave reference sera (data not shown). 58
Table I illustrates the A492 value and P/N ratio frequency distribution of 403 bovine sera against the BVD virus ELISA antigen. The sera were collected from a BVD virus-free herd at ADRI. The mean A492 value was 0.138 + 0.030 and the mean P/N ratio was 1.016 ± 0.102. If a P/N ratio of less than 1.50 was used as the cut-off value for negative, the specificity of this ELISA in 403 BVD virus-free bovine sera was 100%. RELATIVE SENSITIVITY OF ELISA
Two hundred and ninety-six sera which had BVD virus SN antibody titers of > 1:2 were used to determine the relative sensitivity of the present ELISA (Table II). Of these sera, 289 sera were ELISA positive and two sera were ELISA negative (false negative 0.7%), and there were five sera (1.7%) with suspicious reactions. Thus, the relative sensitivity of the present ELISA was 97.6%. KAPPA STATISTICS
Six hundred and ninety-nine sera were used to determine the agreement between ELISA and SN tests. Table III illustrates the Kappa statistics. The TABLE I. Frequency distribution of A492 value and P/N ratio of normal bovine sera (n = 403) against BVD viral (Singer Strain) antigen in the ELISA Absorbance at 492 nm (A492) < 0.074 0.075 - 0.099 0.100-0.124 0.125 -0.149 0.150 - 0.174 0.175-0.199 0.200 - 0.224 > 0.225 Total Mean ± SD = 0.1380 ± 0.30
No. of sera 6 54 54 140 106 38 5 0 403
P/N Ratio 0.70 - 0.89 0.90- 1.09 1.10-1.29 1.30- 1.49 >1.50 Total
No. of sera 41 274 86 2 0 403
Mean±5D= 1.016±0.102
TABLE II. Comparison of ELISA with SN test
ELISAb results Nega- Suspi- Positive cious tive No. SNa titer 24 2 1:2 - 1:10 29 3 2 1:16- 1:40 83 0 81 0 59 1:64- 1:80 59 0 > 1:128 0 125 125 0 2 Total 5 289 296 aSerum neutralization test for BVD virus antibodies bEnzyme-linked immunosorbent assay
ELISA results gave an excellent agreement with SN in detecting both seropositive and seronegative animals. The Kappa value was 0.994 with a 95% confidence limit of 0. 92-1.00.
DISCUSSION In the present study, BVD viral antigen for use in the ELISA was prepared first by solubilizing BVD virus infected cell pellets with the detergent MEGA-10 as described by Juntti et al (5). The inherent problem of high background reactions of the antigen in ELISA was eliminated by further treatment of the solubilized antigen with Triton X-100 and by removing the Triton X-100 bound proteins and free Triton X-100 by centrifugation and adsorption with Bio-beads SM-2. The Triton X series of nonionic detergents has been known to bind preferentially to hydrophobic proteins (10,11). Thus, hydrophobic proteins (bound to Triton X-100), which were the probable cause of nonspecific high background reactions were eliminated or remained masked, and predominantly hydrophilic proteins were enriched in the present BVD viral antigen. Nonionic detergents such as Triton X-100 and Nonidet P40 (NP40) have been shown to retard or prevent protein binding to microtiter wells (1214). Therefore, their removal during antigen preparation is desired. It has been shown previously that there was considerable difficulty with NP40 treated BVD viral antigen in the attachment of the antigen on ELISA plates (2,3). In another study, when NP40 solubilized porcine epidemic diarrhea coronavirus was used for an ELISA, no reactions were detected at low dilutions of antigen and only when antigen dilutions greater than 1:150
TABLE III. Comparison of ELISA and SN tests (n = 699) by a 2 x 2 table and Kappa statistics
SNa Category +
ELISAb
_
+
-
294c 2
0 403
Sum 294 405
699 296 403 Sum = 0.997 Agreement observed = 0.512 Agreement due to chance = 0.994 Kappa = 0.038 SE (K) = 0.92 - 1.00 95% Confidence limits (K) = 26.28 Kappa/ SE(K) aSerum neutralization test for BVD virus
antibodies bEnzyme-linked immunosorbent assay cELISA suspicious reactions (N = 5) were considered as ELISA positive category in calculating the Kappa statistics
were used was an optical density of 0.9 reached (14). As seen in Fig. 2, the present BVD viral antigen did not cause high background reaction problems and readily attached to the microtiter plates and thus enabled us to detect BVD viral antibody in bovine sera. Based on the results obtained, the ELISA method described here appears to be an efficient serological test in terms of excellent specificity and sensitivity. It agrees closely with the SN test in detecting both seropositive and seronegative animals (Kappa = 0.994). The test is also more practical and faster than the SN test for the detection of BVD viral antibody. Because of its many advantages over the SN test, such as its convenient technical features, together with its
potential adaptability for automation, this ELISA method may have value as an alternative test for the detection of BVD viral antibodies in cattle. ACKNOWLEDGMENTS The authors thank Mr. P. Mohanraj for his excellent assistance with the ELISA and Mrs. S.E. Smithson and Mr. W.A. Pickering for assistance with cell culture and serum neutralization tests. Drs. V.W. Lees and K.G. Loewen supplied many of the serum samples used in this study and reference positive sera. Dr. V.W. Lees also gave valuable advice on Kappa statistics. We are also grateful to Ms. S.M. Braun for preparing the figures and Miss S. Van Dyk for assistance in the manuscript preparation.
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