Veterinary Microbiology, 21 (1990) 275-281 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
275
B l o c k i n g E n z y m e - L i n k e d Immunosorbent Assay for Detection of Antibodies Against Bovine Enterovirus A.Q. ZHANG*, J.R. SMITH and G.W. BURGESS
Graduate School of Tropical Veterinary Science, James Cook University, TownsviUe, Qld. 4811 (Australia) (Accepted for publication 21 July 1989)
ABSTRACT Zhang, A.Q., Smith, J.R. and Burgess, G.W., 1990. Blocking enzyme-linkedimmunosorbentassay for detection of antibodies against bovine enterovirus. Vet. Microbiol., 21: 275-281. A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against bovine enterovirus (BEV) in bovine sera. In this ELISA, bovine serum samples were allowed to react with captured viral antigens (by specific chicken IgG), before the addition of specific mouse IgG for measuring non-occupied viral epitopes. The ELISA was slightly more sensitive and required a shorter time period than traditional serum neutralisation (SN). Among the 871 bovine serum samples tested so far, the titres produced by this assay had a significant correlation with those recorded by SN. The ELISA could be used as an alternative assay for SN in a large-scale BEV antibody investigation.
INTRODUCTION
Bovine enterovirus (BEV) causes diarrhoea in calves (Dunne et al., 1974). During the infection, the specific viral antigens can be detected in the epithelial cells of the entire intestinal tract, as well as in the cells of the reticuloendothelial system and lymphatic system (Fernando, 1974). The virus, in combination with Escherichia coli, causes pneumonia. The pneumonic lesions are more extensive than when E. coli alone is inoculated (Spradbrow and Cole, 1971). BEV can also complicate the eradication of foot-and-mouth disease (FMD). Serological comparison between FMD virus (FMDV) and BEV shows the common epitopes shared by the two viruses (Andersen, 1978). Penetration of FMDV RNA into the BEV capsid makes the control of FMD much more difficult (Trautman and Sutmoller, 1971 ). *Present address: Center for Advanced Biotechnology and Medicine, 675 Hoes Lane, Piscataway, NJ 08854, U.S.A.
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
276
A.Q. ZHANG ET AL.
Traditionally, specific BEV antibodies are detected by the immunodiffusion test (Andersen, 1978) and serum neutralisation (SN) test (Rovozzo and Luginbuhl, 1965). However, these tests have several disadvantages. The immunodiffusion test lacks sensitivity, while SN is costly and time consuming. In this report, we describe a blocking enzyme-linked immunosorbent assay (ELISA) for the detection of specific antibodies against BEV. MATERIALSAND METHODS
Virus and cell culture BEV strain K2577 was isolated in Queensland (Zhang and Burgess, 1986). The virus was inoculated on to Madin-Darby bovine kidney (MDBK) cell monolayers at a multiplicity of infection of 10 median cell culture infective doses (CCIDso). After 1 h of adsorption, the infected cells were washed with Dulbecco's modified Eagle's medium (DMEM) and incubated in serum-free DMEM at 37 °C in a 5% CO2 atmosphere. When the cytopathic effect (CPE) was fully developed in the cell monolayers, usually within 15 h post-infection (Zhang et al., 1988), the infected cell cultures were frozen at - 2 0 ° C. After thawing, the viral suspensions were concentrated to 100:1 by 50% (v/v) ammonium sulphate precipitation (Brown and Cartwright, 1963).
Viral purification Viral suspensions were clarified by an initial centrifugation at 1000 Xg for 10 min, followed by centrifugation at 10 000 Xg for 30 min. Virus was pelleted by ultracentrifugation at 100 000Xg (Type 70 Ti. 1, Beckman) for 120 min. The resuspended virus was purified on a linear 15-45% (w/v) sucrose gradient in 100 mM Tris and 100 m M NaC1 buffer by centrifugation at 80 000 x g for 240 min. One percent sodium dodecyl sulphate was added to viral samples before sucrose gradient centrifugation, as suggested for picornavirus purification (Brown et al., 1976 ). Fractions containing virions were detected by infectivity and pooled.
Preparation of catching and indicating antibodies A chicken and a mouse were particularly selected for antiserum production because the non-specific 7-globulin interaction between the two animals was minimal. The animals were inoculated four times with 107 CCIDso of purified virions at intervals of 6 weeks. The first three injections were given subcutaneously and intramuscularly (or intraperitoneally for mouse) at multiple sites, and the inoculum was mixed with an equal volume of Freund's complete adjuvant (Commonwealth Serum Laboratories, Australia) for the first injection
BLOCKING ELISA FOR BOVINE ENTEROVIRUS ANTIBODIES
277
and Freund's incomplete adjuvant (Commonwealth Serum Laboratories, Australia) for the second and third injections. The last injection was given intravenously without using adjuvant. The antisera were collected 10 days after the last injection. Chicken antiserum ~-globulins were precipitated three times in 35% (v/v) ammonium sulphate (Hebert, 1974). Mouse antiserum ~-globulins were precipitated once in 35% (v/v) ammonium sulphate and twice in 40% (v/v) ammonium sulphate (Hebert, 1974). Immunoglobulin G (IgG) of the ~-globulin suspensions were purified by AFFI-GEL-BLUE chromatography (Bio-Rad, CA). Preparation of bovine sera Hyperimmune calf sera Six calves (3-6 months old) were inoculated intravenously twice with 108 CCIDso of BEV at an interval of 45 days. The sera were collected 10 days after the second injection. Bovine foetal sera Two bovine foetal sera were purchased (Commonwealth Serum Laboratories, Australia). The sera were tested in SN against BEV. No neutralising activity against BEV was found in the sera. The two sera were used as nonreactive sera in the following ELISA. Bovine serum samples A total of 871 cattle sera were randomly collected from every state in Australia. Serum neutralisation Before the test, each bovine serum was inactivated at 56 oC for 30 min. Fifty microlitres of viral suspension containing 100 CCIDso of BEV were mixed with 50 ~l of 2-fold dilutions of serum samples in DMEM in microtitre plates for 1 h. The serum samples were tested in triplicate. Then, 50/~l of MDBK cells (20 000 cells per 50/11) in DMEM with 6% normal bovine foetal serum were added to each well. The plates were sealed and incubated at 37°C until full CPE appeared. Blocking E L I S A Fifty microlitres per well of the hyperimm~ne chicken IgG against the virus, at a dilution of 2.5/~g m1-1 in PBS, were coated on to polyvinyl chloride plates overnight at 22 °C as catching antibody. The plates were washed three times in phosphate-buffered saline containing 0.05% Tween 20 (PBST). Then, 50
278
A.Q. ZHANG ET AL.
/tl per well of virus-infected cell culture supernatant, containing 105 CCIDs0 of virus in P B S T , were added. One hour later, the plates were washed and 50 gl per well of bovine test serum samples at a dilution of I : 100 in P B S T , containing 1% ( v / v ) normal chicken serum, 1% ( w / v ) lysine, 1% ( w / v ) glycine and 1% ( w / v ) glutamine, were added. After 1 h incubation, the plates were washed three times in P B S T . Fifty microlitres per well of the hyperimmune mouse IgG against the virus at a dilution of 5/tg m1-1 in P B S T , containing 5% ( v / v ) normal bovine foetal serum, were added as indicating antibody. One hour later, the plates were washed; 50 ]~l per well of goat anti-mouse IgG conjugated with peroxidase (affinity purified product, Biorad, CA) at a dilution of 1:3000 in P B S T , containing 0.5 mg ml-1 casein, were added. After 1 h incubation, the plates were washed; 100 gl of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) substrate solution ( 1 m M ) was added to each well. One hour later, optical density (OD) values were read at a wavelength of 410 nm. A standard titration curve using a hyperimmune calf serum (No. 5125 ) was constructed. A zero blocking line was drawn at the OD value where no test bovine serum was added. A 50% blocking line was drawn at half of the OD value. The 50% blocking line always crosses the exponential phase of the titration curve. It was arbitrarily decided that the sixth dilution over the 50% blocking line was an endpoint in this blocking ELISA. Based on this endpoint, any OD values could be expressed as reciprocal antibody titres simply by comparing OD values of samples with the titration curve. RESULTS
Antibody detection in blocking ELISA The E L I S A produced high titres to the six hyperimmune calf sera (titre range 500-5000 ), but no titres to the selected non-reactive bovine foetal sera. The other 871 bovine serum samples had various titres, as shown in Fig. 1.
Agreement between S N and blocking ELISA If sera with S N titres < 6 or with E L I S A titres < 16 were arbitrarily regarded as non-reactive sera, the agreement between the two assays was 85% (740/ 871). There were 64 sera (7.3%) regarded as reactive sera in the blocking ELISA, but as non-reactive sera in SN. There were 67 sera (7.7%) regarded as non-reactive sera in ELISA, b u t as reactive sera in SN. Among the 871 test bovine serum samples, titres produced by blocking ELISA had a linear relationship with those recorded by SN. The results are shown in Fig. 1. The correlation between the two assays was very significant (r--0.6577, P
275
B l o c k i n g E n z y m e - L i n k e d Immunosorbent Assay for Detection of Antibodies Against Bovine Enterovirus A.Q. ZHANG*, J.R. SMITH and G.W. BURGESS
Graduate School of Tropical Veterinary Science, James Cook University, TownsviUe, Qld. 4811 (Australia) (Accepted for publication 21 July 1989)
ABSTRACT Zhang, A.Q., Smith, J.R. and Burgess, G.W., 1990. Blocking enzyme-linkedimmunosorbentassay for detection of antibodies against bovine enterovirus. Vet. Microbiol., 21: 275-281. A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against bovine enterovirus (BEV) in bovine sera. In this ELISA, bovine serum samples were allowed to react with captured viral antigens (by specific chicken IgG), before the addition of specific mouse IgG for measuring non-occupied viral epitopes. The ELISA was slightly more sensitive and required a shorter time period than traditional serum neutralisation (SN). Among the 871 bovine serum samples tested so far, the titres produced by this assay had a significant correlation with those recorded by SN. The ELISA could be used as an alternative assay for SN in a large-scale BEV antibody investigation.
INTRODUCTION
Bovine enterovirus (BEV) causes diarrhoea in calves (Dunne et al., 1974). During the infection, the specific viral antigens can be detected in the epithelial cells of the entire intestinal tract, as well as in the cells of the reticuloendothelial system and lymphatic system (Fernando, 1974). The virus, in combination with Escherichia coli, causes pneumonia. The pneumonic lesions are more extensive than when E. coli alone is inoculated (Spradbrow and Cole, 1971). BEV can also complicate the eradication of foot-and-mouth disease (FMD). Serological comparison between FMD virus (FMDV) and BEV shows the common epitopes shared by the two viruses (Andersen, 1978). Penetration of FMDV RNA into the BEV capsid makes the control of FMD much more difficult (Trautman and Sutmoller, 1971 ). *Present address: Center for Advanced Biotechnology and Medicine, 675 Hoes Lane, Piscataway, NJ 08854, U.S.A.
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
276
A.Q. ZHANG ET AL.
Traditionally, specific BEV antibodies are detected by the immunodiffusion test (Andersen, 1978) and serum neutralisation (SN) test (Rovozzo and Luginbuhl, 1965). However, these tests have several disadvantages. The immunodiffusion test lacks sensitivity, while SN is costly and time consuming. In this report, we describe a blocking enzyme-linked immunosorbent assay (ELISA) for the detection of specific antibodies against BEV. MATERIALSAND METHODS
Virus and cell culture BEV strain K2577 was isolated in Queensland (Zhang and Burgess, 1986). The virus was inoculated on to Madin-Darby bovine kidney (MDBK) cell monolayers at a multiplicity of infection of 10 median cell culture infective doses (CCIDso). After 1 h of adsorption, the infected cells were washed with Dulbecco's modified Eagle's medium (DMEM) and incubated in serum-free DMEM at 37 °C in a 5% CO2 atmosphere. When the cytopathic effect (CPE) was fully developed in the cell monolayers, usually within 15 h post-infection (Zhang et al., 1988), the infected cell cultures were frozen at - 2 0 ° C. After thawing, the viral suspensions were concentrated to 100:1 by 50% (v/v) ammonium sulphate precipitation (Brown and Cartwright, 1963).
Viral purification Viral suspensions were clarified by an initial centrifugation at 1000 Xg for 10 min, followed by centrifugation at 10 000 Xg for 30 min. Virus was pelleted by ultracentrifugation at 100 000Xg (Type 70 Ti. 1, Beckman) for 120 min. The resuspended virus was purified on a linear 15-45% (w/v) sucrose gradient in 100 mM Tris and 100 m M NaC1 buffer by centrifugation at 80 000 x g for 240 min. One percent sodium dodecyl sulphate was added to viral samples before sucrose gradient centrifugation, as suggested for picornavirus purification (Brown et al., 1976 ). Fractions containing virions were detected by infectivity and pooled.
Preparation of catching and indicating antibodies A chicken and a mouse were particularly selected for antiserum production because the non-specific 7-globulin interaction between the two animals was minimal. The animals were inoculated four times with 107 CCIDso of purified virions at intervals of 6 weeks. The first three injections were given subcutaneously and intramuscularly (or intraperitoneally for mouse) at multiple sites, and the inoculum was mixed with an equal volume of Freund's complete adjuvant (Commonwealth Serum Laboratories, Australia) for the first injection
BLOCKING ELISA FOR BOVINE ENTEROVIRUS ANTIBODIES
277
and Freund's incomplete adjuvant (Commonwealth Serum Laboratories, Australia) for the second and third injections. The last injection was given intravenously without using adjuvant. The antisera were collected 10 days after the last injection. Chicken antiserum ~-globulins were precipitated three times in 35% (v/v) ammonium sulphate (Hebert, 1974). Mouse antiserum ~-globulins were precipitated once in 35% (v/v) ammonium sulphate and twice in 40% (v/v) ammonium sulphate (Hebert, 1974). Immunoglobulin G (IgG) of the ~-globulin suspensions were purified by AFFI-GEL-BLUE chromatography (Bio-Rad, CA). Preparation of bovine sera Hyperimmune calf sera Six calves (3-6 months old) were inoculated intravenously twice with 108 CCIDso of BEV at an interval of 45 days. The sera were collected 10 days after the second injection. Bovine foetal sera Two bovine foetal sera were purchased (Commonwealth Serum Laboratories, Australia). The sera were tested in SN against BEV. No neutralising activity against BEV was found in the sera. The two sera were used as nonreactive sera in the following ELISA. Bovine serum samples A total of 871 cattle sera were randomly collected from every state in Australia. Serum neutralisation Before the test, each bovine serum was inactivated at 56 oC for 30 min. Fifty microlitres of viral suspension containing 100 CCIDso of BEV were mixed with 50 ~l of 2-fold dilutions of serum samples in DMEM in microtitre plates for 1 h. The serum samples were tested in triplicate. Then, 50/~l of MDBK cells (20 000 cells per 50/11) in DMEM with 6% normal bovine foetal serum were added to each well. The plates were sealed and incubated at 37°C until full CPE appeared. Blocking E L I S A Fifty microlitres per well of the hyperimm~ne chicken IgG against the virus, at a dilution of 2.5/~g m1-1 in PBS, were coated on to polyvinyl chloride plates overnight at 22 °C as catching antibody. The plates were washed three times in phosphate-buffered saline containing 0.05% Tween 20 (PBST). Then, 50
278
A.Q. ZHANG ET AL.
/tl per well of virus-infected cell culture supernatant, containing 105 CCIDs0 of virus in P B S T , were added. One hour later, the plates were washed and 50 gl per well of bovine test serum samples at a dilution of I : 100 in P B S T , containing 1% ( v / v ) normal chicken serum, 1% ( w / v ) lysine, 1% ( w / v ) glycine and 1% ( w / v ) glutamine, were added. After 1 h incubation, the plates were washed three times in P B S T . Fifty microlitres per well of the hyperimmune mouse IgG against the virus at a dilution of 5/tg m1-1 in P B S T , containing 5% ( v / v ) normal bovine foetal serum, were added as indicating antibody. One hour later, the plates were washed; 50 ]~l per well of goat anti-mouse IgG conjugated with peroxidase (affinity purified product, Biorad, CA) at a dilution of 1:3000 in P B S T , containing 0.5 mg ml-1 casein, were added. After 1 h incubation, the plates were washed; 100 gl of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) substrate solution ( 1 m M ) was added to each well. One hour later, optical density (OD) values were read at a wavelength of 410 nm. A standard titration curve using a hyperimmune calf serum (No. 5125 ) was constructed. A zero blocking line was drawn at the OD value where no test bovine serum was added. A 50% blocking line was drawn at half of the OD value. The 50% blocking line always crosses the exponential phase of the titration curve. It was arbitrarily decided that the sixth dilution over the 50% blocking line was an endpoint in this blocking ELISA. Based on this endpoint, any OD values could be expressed as reciprocal antibody titres simply by comparing OD values of samples with the titration curve. RESULTS
Antibody detection in blocking ELISA The E L I S A produced high titres to the six hyperimmune calf sera (titre range 500-5000 ), but no titres to the selected non-reactive bovine foetal sera. The other 871 bovine serum samples had various titres, as shown in Fig. 1.
Agreement between S N and blocking ELISA If sera with S N titres < 6 or with E L I S A titres < 16 were arbitrarily regarded as non-reactive sera, the agreement between the two assays was 85% (740/ 871). There were 64 sera (7.3%) regarded as reactive sera in the blocking ELISA, but as non-reactive sera in SN. There were 67 sera (7.7%) regarded as non-reactive sera in ELISA, b u t as reactive sera in SN. Among the 871 test bovine serum samples, titres produced by blocking ELISA had a linear relationship with those recorded by SN. The results are shown in Fig. 1. The correlation between the two assays was very significant (r--0.6577, P
