Journal of Virological Methods,
34 (1991) 181-192 0 1991 Elsevier Science Publishers B.V. / All rights reserved/0166-0934/91/%03.50 ADONIS 016609349100428L
181
VIRMET 01221
The detection of antibodies against foot-and-mouth disease virus (FMDV) in filter paper eluates from pig sera or whole blood by ELISA R.M. Armstrong, J.R. Crowther and M.S. Denyer A.F.R.C.
Institute for Animal Health, Pirbright,
U.K.
(Accepted 28 May, 1991)
Summary
The use of pig blood samples dried on paper discs for the detection of antibodies against FMDV by the liquid phase blocking ELISA has been evaluated. The average volume of whole heparinised blood required to fully saturate 6.0 mm discs was 7.65 ~1 (range 7.2 to 8.1; variation = 0.24, P = 0.05). When 200 clinically healthy animals were assessed by virus neutralisation (VN) titres up to l/22 were recorded against types 0, A and C, 97% being l/l 1 or less. Using ELISA, results were more skewed. Overall, 91% showed titres of l/32 or less, and there were occasional high non-specific reactors with types 0 and A. Using small groups of sera from experimental animals, a VN titre of l/ 16 was found to be equivalent to an ELISA titre of approximately l/100 (logic 2.0 + 0.2) with type 0 and l/56 (logi,-, 1.75 + 0.1) for type A. Although some loss in sensitivity from disc dried sera was found in selected negatives on testing at a single dilution of l/32, sera from vaccinated animals with VN titres of l/16 to l/708 all gave strong positive results. A monoclonal antibody (Mab) assay was successfully developed to detect different swine anti-FMDV antibody isotypes. ELISA; Eluate; Foot-and-mouth
Correspondence
to:
Woking, Surrey, U.K.
R.M. Armstrong,
disease virus; Antibody; Isotype
A.F.R.C. Institute for Animal Health, Ash Road, Pirbright,
182
Introduction
The testing of animals for antibodies against infectious agents before import or export can be a laborious and lengthy process. Swine can be particularly difficult to bleed and may require shackling. Samples sent as whole clotted blood may deteriorate if their arrival at the laboratory is delayed and breakage, with the subsequent leakage of fluid, could present a disease security hazard. In the laboratory, pig sera can exhibit adverse effects, such as toxicity towards cells used in the VN test and the occurrence of high non-specific titres against FMDV (Andersen, 1977). The use of paper discs upon which blood may be dried can greatly simplify sampling and transport of blood to the laboratory. The veins in the ear may be nicked with a scalpel while the animal is feeding and blood allowed to soak onto the paper, saturating the peripheral discs. After drying, the papers can then be delivered to the laboratory. This sampling technique has been combined with ELZSA for the detection of antibodies against FMDV (Gaggero and Sutmoller, 1945; Viera and Fernandes, 1967), swine vesicular disease virusSVDV- (Hamblin and Hedger, 1982) and Aujeszky’s disease virus (Banks, 1985; Motha et al., 1987) with great success as a surveillance tool. The liquid phase blocking ELISA (Hamblin et al. 1986a,b) has now largely replaced the VN test for the routine testing of sera for antibodies against FMDV in this laboratory. It has gained wide acceptance inte~ationally because of its greater sensitivity and it also circumvents the need for tissue culture on a daily basis. The assay is very simple to perform and is extremely economical with reagents, particularly test sera. Monoclonal antibodies are powerful tools which can be used to give greater insight into an antibody response to infection or vaccination (Gooding, 1986). In this paper, we have described the work performed in adapting the paper sampling method for use in ELISA and the development of an assay to detect swine antibody isotypes against FMDV.
Materials and Methods
Quadrant shaped Whatman (Maidstone, Kent, England) wet strengthened paper (no. 113) with six peripheral discs of approximately 6.0 mm in diameter attached, were obtained from the Sales Dispatch Unit, Central Veterinary Laboratory, Weybridge, U.K. KTl5 3NB. Virus strains
The FMDV strains OiBFS 1860 (U.K. 1967), A5 Parma (Italy, 1962), AS AlIier (France, 1960/FRA l/68), and C Noville (Switzerland, 1965) were grown in BHK-21 cells.
183
Negative swine sera Approximately 200 negative pig sera were from clinically healthy animals, freshly submitted for testing prior to export from the U.K. in Autumn 1990. They were from a wide range of geo~aphi~l locations and, where possible, all the sera in a given batch were tested. Initial testing was carried out within two weeks of receipt. Whole blood from ten swine kept in isolation at the Institute for Animal Health (IAH), Pirbright was collected in Lithium Heparin vacutainers (Becton Dickinson, Cowley, U.K.) and stored at + 4°C or - 70°C. Positive anti-FMDV pig sera and whole blood Two separate batches of positive sera were used. The first were from experimental swine vaccinated and/or challenged with AS Parma in isolation at IAH, Pirbright. Whole blood samples were also collected from these animals as described above. The second group were taken from pigs vaccinated with OlBFS 1860 and were obtained from Pitman-Moore U.K. Ltd., U.K. Rabbit and guinea-pig anti-FMD V sera Hyperimmune rabbit anti-FMDV sera were produced according to the method of Have et al. (1984) while the method described by Ferris and Donaldson (1984) was used for guinea-pig sera. For use in Mab assays, rabbit anti-A5 Parma sera was added to an equal volume of bovine serum agarose (Sigma, Dorset, U.K.) and shaken at 37°C for 1 h. It was then centrifuged at 3000 rpm on a bench centrifuge for 5 min and the supernatant was carefully aspirated. The absorbed serum was used at its optimal dilution, dete~ined by checkerboard titration. Virus neutralisation tests The testing of negative and positive pig sera was performed in flat-bottomed tissue culture grade microtitre plates (Golding et al., 1976) using 1%RS-2 cells at a concentration of lo6 cells per ml (see Fig. 2). Viruses and sera were diluted in Eagle’s MEM containing 30 mm01 HEPES buffer. Cells were initially suspended in Eagle’s MEM containing 10% adult bovine serum. Plates were read microscopically from 20 to 68 h. A well was considered positive if any cytopathic effect was observed. Antibody titres were expressed as the reciprocal of the final dilution of serum giving 50% neutralisation in the presence of approximately 100 TCIDso virus. ~onoclona~ ~tibodies (Mabs) Ascites fluids containing Mabs against swine IgA (K61 lB4), lgG* and IgG2 (K1384C12) and IgM (K513Dl) were kindly supplied by C. Stokes and K.
184
Stevens, of School of Veterinary ELISA
Science, University
of Bristol, Bristol, U.K.
diluent
Filtered phosphate buffered saline pH 7.6 (PBS) containing 0.05% Tween 20 and phenol red (PBST) was used for the liquid phase blocking ELISA while PBS with 1% Tween 80 (Ohkubo et al. 1984), 3% bovine serum albumin, 5% adult bovine serum and 1% NaCl (PBST80) was used for Mab assays. ELISA
washing fluid
Filtered phosphate buffered saline pH 7.6 (PBS) was used for the blocking ELISA while PBST without phenol red was used for Mab assays. Determination
of disc saturation
volume
Ten heparinised whole blood samples from ‘clean’ (uninfected) pigs from isolation at IAH, Pirbright were used to assess the volume required to fully saturate a 6.0 mm disc. The stock blood samples were stored at +4”C but moved to room temperature about 2 h before testing (22-24°C). Using a calibrated Eppendorf Varipette 4710 and a new tip for each sample, different volumes of mixed whole blood were applied to the outside of discs in triplicate. The volume of each blood needed to saturate the discs was noted on ten separate occasions. Determination
of elution time required when diluting from eluate
Discs upon which 7.5 ~1 of whole blood from experimental pigs had been allowed to dry for 2 h at room temperature, were stored overnight at +4”C. Elution was performed in round well carrier plates static or agitated on a Rotatest shaker (Luckhams Ltd. W. Sussex, U.K.) at both room temperature or 37°C. The assumption was made that whole blood consists of approximately 70% water and extra diluent was added to allow for evaporation. Discs were therefore added to 57.7 ~1 of PBST in the first row of each plate for various time intervals to allow elution of antibody into the liquid phase. Afterwards, 30 ~1 of PBST were added to all other wells on the plate and a two-fold dilution series made by transfer. An equal volume of a constant dilution (determined by prior titration) of virus in PBST was added to each well, thereby resulting in a first final dilution of whole blood of l/16 and l/32 of serum - assuming the serum component to occupy 50% of the whole blood volume. The mixtures were held overnight at +4”C, then 30 ,ul from each well was sampled onto hard Nuncimmuno maxisorp ELISA plates (Life technology, Uxbridge, Middlesex) coated with rabbit anti-As Parma serum (Hamblin et al. 1986a,b) and the assay continued according to this method except that all reagents were dispensed in 30 ~1 volumes. All titres presented in this study are with respect to serum content.
185 TABLE 1 (a) Total anti-As Parma reciprocal antibody titres in vaccinated swine (average of 2 assays). (b) Percentage change in titre from liquid whole to eluted dried blood. (c) Composition by antibody isotype in liquid whole blood
a Animal number
Separated serum
RN3 RN6 RN7 RN8 RN9 RN11 RN17 RM58 RM62 RM63 RM70
341 181 5120 160 3496 2172 3496 3496 124 4096 272
b
c IgA
Liquid
Eluted
Change
whole blood
dried blood
liquid to dry
109 219 10240 309 2896 2172 1748 3072 1078 2896 384
87 87 4248 77 980 1086 1960 1448 512 2048 155
-20% -60% -59% -75% -66% - 50% +12% -53% -53% - 29% - 60%
34 (1991) 181-192 0 1991 Elsevier Science Publishers B.V. / All rights reserved/0166-0934/91/%03.50 ADONIS 016609349100428L
181
VIRMET 01221
The detection of antibodies against foot-and-mouth disease virus (FMDV) in filter paper eluates from pig sera or whole blood by ELISA R.M. Armstrong, J.R. Crowther and M.S. Denyer A.F.R.C.
Institute for Animal Health, Pirbright,
U.K.
(Accepted 28 May, 1991)
Summary
The use of pig blood samples dried on paper discs for the detection of antibodies against FMDV by the liquid phase blocking ELISA has been evaluated. The average volume of whole heparinised blood required to fully saturate 6.0 mm discs was 7.65 ~1 (range 7.2 to 8.1; variation = 0.24, P = 0.05). When 200 clinically healthy animals were assessed by virus neutralisation (VN) titres up to l/22 were recorded against types 0, A and C, 97% being l/l 1 or less. Using ELISA, results were more skewed. Overall, 91% showed titres of l/32 or less, and there were occasional high non-specific reactors with types 0 and A. Using small groups of sera from experimental animals, a VN titre of l/ 16 was found to be equivalent to an ELISA titre of approximately l/100 (logic 2.0 + 0.2) with type 0 and l/56 (logi,-, 1.75 + 0.1) for type A. Although some loss in sensitivity from disc dried sera was found in selected negatives on testing at a single dilution of l/32, sera from vaccinated animals with VN titres of l/16 to l/708 all gave strong positive results. A monoclonal antibody (Mab) assay was successfully developed to detect different swine anti-FMDV antibody isotypes. ELISA; Eluate; Foot-and-mouth
Correspondence
to:
Woking, Surrey, U.K.
R.M. Armstrong,
disease virus; Antibody; Isotype
A.F.R.C. Institute for Animal Health, Ash Road, Pirbright,
182
Introduction
The testing of animals for antibodies against infectious agents before import or export can be a laborious and lengthy process. Swine can be particularly difficult to bleed and may require shackling. Samples sent as whole clotted blood may deteriorate if their arrival at the laboratory is delayed and breakage, with the subsequent leakage of fluid, could present a disease security hazard. In the laboratory, pig sera can exhibit adverse effects, such as toxicity towards cells used in the VN test and the occurrence of high non-specific titres against FMDV (Andersen, 1977). The use of paper discs upon which blood may be dried can greatly simplify sampling and transport of blood to the laboratory. The veins in the ear may be nicked with a scalpel while the animal is feeding and blood allowed to soak onto the paper, saturating the peripheral discs. After drying, the papers can then be delivered to the laboratory. This sampling technique has been combined with ELZSA for the detection of antibodies against FMDV (Gaggero and Sutmoller, 1945; Viera and Fernandes, 1967), swine vesicular disease virusSVDV- (Hamblin and Hedger, 1982) and Aujeszky’s disease virus (Banks, 1985; Motha et al., 1987) with great success as a surveillance tool. The liquid phase blocking ELISA (Hamblin et al. 1986a,b) has now largely replaced the VN test for the routine testing of sera for antibodies against FMDV in this laboratory. It has gained wide acceptance inte~ationally because of its greater sensitivity and it also circumvents the need for tissue culture on a daily basis. The assay is very simple to perform and is extremely economical with reagents, particularly test sera. Monoclonal antibodies are powerful tools which can be used to give greater insight into an antibody response to infection or vaccination (Gooding, 1986). In this paper, we have described the work performed in adapting the paper sampling method for use in ELISA and the development of an assay to detect swine antibody isotypes against FMDV.
Materials and Methods
Quadrant shaped Whatman (Maidstone, Kent, England) wet strengthened paper (no. 113) with six peripheral discs of approximately 6.0 mm in diameter attached, were obtained from the Sales Dispatch Unit, Central Veterinary Laboratory, Weybridge, U.K. KTl5 3NB. Virus strains
The FMDV strains OiBFS 1860 (U.K. 1967), A5 Parma (Italy, 1962), AS AlIier (France, 1960/FRA l/68), and C Noville (Switzerland, 1965) were grown in BHK-21 cells.
183
Negative swine sera Approximately 200 negative pig sera were from clinically healthy animals, freshly submitted for testing prior to export from the U.K. in Autumn 1990. They were from a wide range of geo~aphi~l locations and, where possible, all the sera in a given batch were tested. Initial testing was carried out within two weeks of receipt. Whole blood from ten swine kept in isolation at the Institute for Animal Health (IAH), Pirbright was collected in Lithium Heparin vacutainers (Becton Dickinson, Cowley, U.K.) and stored at + 4°C or - 70°C. Positive anti-FMDV pig sera and whole blood Two separate batches of positive sera were used. The first were from experimental swine vaccinated and/or challenged with AS Parma in isolation at IAH, Pirbright. Whole blood samples were also collected from these animals as described above. The second group were taken from pigs vaccinated with OlBFS 1860 and were obtained from Pitman-Moore U.K. Ltd., U.K. Rabbit and guinea-pig anti-FMD V sera Hyperimmune rabbit anti-FMDV sera were produced according to the method of Have et al. (1984) while the method described by Ferris and Donaldson (1984) was used for guinea-pig sera. For use in Mab assays, rabbit anti-A5 Parma sera was added to an equal volume of bovine serum agarose (Sigma, Dorset, U.K.) and shaken at 37°C for 1 h. It was then centrifuged at 3000 rpm on a bench centrifuge for 5 min and the supernatant was carefully aspirated. The absorbed serum was used at its optimal dilution, dete~ined by checkerboard titration. Virus neutralisation tests The testing of negative and positive pig sera was performed in flat-bottomed tissue culture grade microtitre plates (Golding et al., 1976) using 1%RS-2 cells at a concentration of lo6 cells per ml (see Fig. 2). Viruses and sera were diluted in Eagle’s MEM containing 30 mm01 HEPES buffer. Cells were initially suspended in Eagle’s MEM containing 10% adult bovine serum. Plates were read microscopically from 20 to 68 h. A well was considered positive if any cytopathic effect was observed. Antibody titres were expressed as the reciprocal of the final dilution of serum giving 50% neutralisation in the presence of approximately 100 TCIDso virus. ~onoclona~ ~tibodies (Mabs) Ascites fluids containing Mabs against swine IgA (K61 lB4), lgG* and IgG2 (K1384C12) and IgM (K513Dl) were kindly supplied by C. Stokes and K.
184
Stevens, of School of Veterinary ELISA
Science, University
of Bristol, Bristol, U.K.
diluent
Filtered phosphate buffered saline pH 7.6 (PBS) containing 0.05% Tween 20 and phenol red (PBST) was used for the liquid phase blocking ELISA while PBS with 1% Tween 80 (Ohkubo et al. 1984), 3% bovine serum albumin, 5% adult bovine serum and 1% NaCl (PBST80) was used for Mab assays. ELISA
washing fluid
Filtered phosphate buffered saline pH 7.6 (PBS) was used for the blocking ELISA while PBST without phenol red was used for Mab assays. Determination
of disc saturation
volume
Ten heparinised whole blood samples from ‘clean’ (uninfected) pigs from isolation at IAH, Pirbright were used to assess the volume required to fully saturate a 6.0 mm disc. The stock blood samples were stored at +4”C but moved to room temperature about 2 h before testing (22-24°C). Using a calibrated Eppendorf Varipette 4710 and a new tip for each sample, different volumes of mixed whole blood were applied to the outside of discs in triplicate. The volume of each blood needed to saturate the discs was noted on ten separate occasions. Determination
of elution time required when diluting from eluate
Discs upon which 7.5 ~1 of whole blood from experimental pigs had been allowed to dry for 2 h at room temperature, were stored overnight at +4”C. Elution was performed in round well carrier plates static or agitated on a Rotatest shaker (Luckhams Ltd. W. Sussex, U.K.) at both room temperature or 37°C. The assumption was made that whole blood consists of approximately 70% water and extra diluent was added to allow for evaporation. Discs were therefore added to 57.7 ~1 of PBST in the first row of each plate for various time intervals to allow elution of antibody into the liquid phase. Afterwards, 30 ~1 of PBST were added to all other wells on the plate and a two-fold dilution series made by transfer. An equal volume of a constant dilution (determined by prior titration) of virus in PBST was added to each well, thereby resulting in a first final dilution of whole blood of l/16 and l/32 of serum - assuming the serum component to occupy 50% of the whole blood volume. The mixtures were held overnight at +4”C, then 30 ,ul from each well was sampled onto hard Nuncimmuno maxisorp ELISA plates (Life technology, Uxbridge, Middlesex) coated with rabbit anti-As Parma serum (Hamblin et al. 1986a,b) and the assay continued according to this method except that all reagents were dispensed in 30 ~1 volumes. All titres presented in this study are with respect to serum content.
185 TABLE 1 (a) Total anti-As Parma reciprocal antibody titres in vaccinated swine (average of 2 assays). (b) Percentage change in titre from liquid whole to eluted dried blood. (c) Composition by antibody isotype in liquid whole blood
a Animal number
Separated serum
RN3 RN6 RN7 RN8 RN9 RN11 RN17 RM58 RM62 RM63 RM70
341 181 5120 160 3496 2172 3496 3496 124 4096 272
b
c IgA
Liquid
Eluted
Change
whole blood
dried blood
liquid to dry
109 219 10240 309 2896 2172 1748 3072 1078 2896 384
87 87 4248 77 980 1086 1960 1448 512 2048 155
-20% -60% -59% -75% -66% - 50% +12% -53% -53% - 29% - 60%
