Veterinary Microbiology, 32 (1992) 253-265 Elsevier Science Publishers B.V., Amsterdam
253
Foot-and-mouth disease: detection of antibodies in cattle sera by blocking ELISA K.J. Sorensen, R.L. Madekurozwa and P. Dawe Veterinary Research Laboratory, Harare, Zimbabwe (Accepted 13 February 1992)
ABSTRACT Sorensen, K.J., Madekurozwa, R.L. and Dawe, P., 1992. Foot-and-mouth disease: detection of antibodies in cattle sera by blocking ELISA. Vet. Microbiol., 32: 253-265.
A blocking ELISA was developed for the detection of antibodies to foot-and-mouth disease virus SAT1, SAT2 and SAT3 and for the quantification of antibodies on a single dilution of serum. The avidin-biotin system was used. The test was compared with the liquid-phase ELISA executed at the World Reference Laboratory for foot-and-mouth disease. It was found to have favourable logistics and combined high specificity with high sensitivity. The quantitative test using a single dilution of serum was resource saving and proved to be a reliable and precise method for the assessment of antibody levels.
INTRODUCTION
The control of foot-and-mouth disease ( F M D ) is of major importance to Zimbabwe in particular because of exports of animals and animal produce. As a result o f the economic implications of the disease major resources are invested in its control. The confinement of the buffalo herds to the national parks and hunting areas and their segregation from the domestic cattle is sustained by hundreds of kilometers of game fences. Vaccinations of cattle herds are carried out twice a year in zones immediately adjacent to the national parks. Buffer zones are maintained between the vaccination zones and the FMD free area. The division into zones is sustained by thousands of kilometers of cattle fences and cattle movement between and out of the zones is controlled. Large scale serological examinations are needed to ascertain the freedom from infection or vaccination of the cattle in the buffer zones and in the FMD free area and also to assess antibody levels in the vaccinated cattle. It therefore became necessary to introduce an ELISA which would give high Correspondence to: K.J. Sorensen, Veterinary Research Laboratory, P.O. Box 8101, Causeway, Harare, Zimbabwe.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
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sensitivity, high specificity and precision. As the veterinary Research Laboratory (VRL) in Harare had no security facilities for handling live FMD virus it also had to be a safe test. In addition the concern for logistics and for keeping expenditure as low as possible required it to be simple and give a high output. Liquid phase (LP)-ELISA was found to be sensitive ( Hamblin et al., 1986a ) but the Department of Veterinary Services in Zimbabwe had experienced problems with the specificity of the test (Hargreaves, pers. c o m m u n . ) . The cost of the test is high because it requires serial dilution of the samples and for the use of 2 different types of plates for each test. Blocking ELISA has previously been applied to FMD control (Have and Holm Jensen, 1983; Westbury et al., 1988). It has been used with great efficiency in the control and eradication of other diseases such as Aujeszky's disease (Sorensen and Lei, 1986; Sorensen et al., 1986) and enzootic bovine leucosis (Hof Jorgensen, 1989) and has proved to unite precision, high sensitivity and specificity with relative simplicity and low cost. Therefore a solid phase blocking ELISA was developed using inactivated virus produced at Botswana Vaccine Institute (BVI), Gaborone. This paper describes the test and presents comparative results with the LP-ELISA. MATERIALS AND METHODS
Antigen Crude inactivated FMD virus type SAT 1 BOT1/77, SAT 2 ZIM 7/89 and SAT 3 ZIM 9/81 was used as antigen. The virus was grown on bovine tongue epithelium using the Frenkel technique according to standard procedure at BVI (Guinet et al., 1982 ). It was inactivated with ethylene imine after clarification and filtration (Preaud et al., 1989). Purified inactivated 146 S particles obtained by sucrose gradient centrifugation (Fayet et al., 1969) was used for immunization of rabbits. Immune sera Two rabbits were used for each of the three SAT types. Each rabbit received two inoculations three weeks apart with about 25 #g of 146 S particles emulsified in equal volumes of Freund's complete adjuvant. The rabbits were bled repeatedly from ear veins three to five weeks after the last injection. The collected serum fractions were pooled, sodium azide was added to 0.1% and they were stored at 4 ° C. Isolation of rabbit IgG DEAE Affi-Gel Blue agarose (Bio-Rad, cat. 153-7307) was used to isolate the IgG fraction. The procedure recommended by the producer was applied. Briefly: 1 ml of serum was dialysed overnight at 4°C against 0.02 M KzHPO4,
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pH 8.0, 0.02% NAN3. The dialysed serum was layered on top o f a 5 ml econocolumn (Bio-Rad) which had been filled with DEAE Affi-Gel Blue agarose and prewashed with 2 bed volumes of 0.02 M K2HPO4, pH 8.0, 0.2% NAN3. The IgG was eluted with 2 bed volumes of the latter buffer and all the eluate ( 10 ml) was collected. The resultant IgG suspension was stored at 4 oC.
Biotinylation of rabbit IgG Biotinamidocaproate N-hydroxysuccinimide ester (SIGMA, cat. B2643 ) dissolved in dimethylsulfoxide was used for biotinylation. Initially the IgG solution was dialysed overnight at 4°C against O. 1 M NaHCO3 pH 8.5. The protein concentration was determined by spectrophotometry at a wavelength at 280 n m using an extinction coefficient of 1.4 for IgG. One part of biotinamidocaproate N-hydroxysuccinimide ester was added to eight parts of protein and incubated for 4 hours at room temperature. The solution was dialysed overnight at 4°C against PBS pH 7.2. To the biotinylated IgG suspension was added sodium azide to a concentration of 0.1% and normal bovine serum to a concentration of 20%. It was stored at 4 oC.
Test samples A total of 1497 blood samples from herds within the FMD free area in Mashonaland West province of Zimbabwe (from which the EEC imports beef) were collected over a period of seven months during 1990 at the Cold Storage Commission (CSC) slaughterhouse, Chinhoyi where non-vaccinated and non-diseased cattle were slaughtered. An outbreak of FMD SAT2 had occurred on one farm in the area in 1989. The farm was depopulated and a ring vaccination zone was established. Two outbreaks of F M D SAT2 were recorded in the late seventies but except for that the province had been free of FMD and vaccinations had not been carried out. Outbreaks with SAT 1 and SAT3 have never occurred (Condy, 1979). Serum samples ( n = 2 8 0 ) from cattle in Denmark (DK) and England ( U K ) were kindly supplied from the State veterinary Institute for Virus Research, Lindholm, Denmark and from the Central veterinary Laboratory, Weybridge, England. The above mentioned sera were screened with the blocking ELISA and used for assessing the specificity of the test. Sera were collected from a total of 157 cattle in 3 herds vaccinated with trivalent SAT 1, SAT2 and SAT3 vaccine produced at BVI in a ring vaccination zone instituted in May 1989 on the occasion of an outbreak of FMD SAT2 in the Midlands province. The sera were used for comparative testing with the LP-ELISA and the blocking ELISA. They were also used to derive the correlation between the end point titres and the ODp values obtained on a single dilution of serum with the blocking ELISA. Randomly selected sera from 23 submissions sent to the laboratory for FMD
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testing over a period of 3 months were used to test the correlation between end point titres and single dilution titres.
Normal bovine serum (NBS) Sera were collected from FMD free cattle at the CSC slaughterhouse, Chinhoyi mentioned above and pooled. All the sera were tested individually for antibodies against FMDV SAT 1, SAT2 and SAT3 with negative results before pooling, eliminating the chance of contaminating antibodies.
Blocking ELISA (a) Buffers and substrate: 1. Buffer A: 50 m M NaHCO3, 0.02% NaN3 pH 9.6 2. Buffer B: 15 m M Na2HPO4, 3 m M KH2PO4, 500 m M NaCI, 0.05% Tween 20 pH 7.2.3. Buffer C: buffer B containing 3% normal bovine serum. 4. Buffer D: 34 m M Na2HPO4, 4 m M KH2PO4, 140 m M NaC1, 0.05% Tween 20, 0.02% NAN3, 1% normal rabbit serum pH 7.5.5. Substrate: 0.1% tetramethyl-benzidine TMB (Aldrich Chemical Co. Ltd, cat. 86,033-6) dissolved in absolute ethanol and further diluted 1 : I0 in a buffer consisting of 24 mM citric acid, 51 m M Na2HPO4, 0.025% hydrogen peroxide (30% H202), pH 5.0. (b) Consumables and equipment: 1. Plates: Nunc Immunoplate Maxisorp cat. 4-42404.2. Conjugate: Peroxidase conjugated Avidin, Dakopatt, cat. p347. 3. Microplate washer: Titertec 120. 4. Microplate photometer: Titertek Multiscan. (c) Performance Q(the test: The test was carried out as previously described for Aujeszky's disease (Sorensen and Lei, 1986) with some modifications. Briefly: ( 1 ) Plates were coated overnight at 4°C with the highest dilution of rabbit IgG in buffer A giving m a x i m u m catchment of antigen. (2) Antigen was added at an appropriate dilution in buffer B to give an OD value of about 1.0 and incubated at 37°C for 1 hour. (3) Dilution of test samples in buffer D. One well was used per sample diluted 1 : 10 for screening. Serial dilutions for titre determinations were carried out with one well per dilution step. Incubation was overnight at room temperature (20-24 ° C ). Six wells with NBS, 2 wells with a weak positive and 2 wells with a strong positive control serum all diluted 1:10 in buffer D and 6 wells with buffer D only were included on each plate. The wells with NBS or buffer D served as non-inhibited references (NIR). (4) Biotinylated IgG suspension diluted in buffer C to give an OD value of about 1.0 was added and incubated at 37°C for 1 hour. (5) Conjugate diluted 1:10 000 in buffer C was added and incubated for 30 rain at 37 ° C. (6) Substrate was added and incubated at room temperature for about 10 min. (7) Sulphuric acid 1 M was added to stop the reaction. Hundred microliter volumes were used in all steps. Steps 1 to 5 were followed by a wash of 3 cycles without soak periods except for step 3 where the wash was stopped after the 1st cycle and step 5 where a soak period of 30 s
BLOCKING ELISA FOR DETECTION OF ANTIBODIES TO FMDV
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was included on the 2nd cycle. Buffer B was used for washing. OD readings were carried out at a wavelength of 450 nm. ODp values were calculated using the formula: ODp = (sample OD X 100) / (mean OD NIR) Results of the screenings were read at the ODp30, ODp40 and at the ODp50 levels on samples diluted 1 : 10 with NBS serving as NIR. End point titres were expressed as the reciprocal value of the dilution giving an ODp value of 50 with buffer D serving as NIR. ODp values used for determination oftitres on a single dilution of serum (single dilution titre) were calculated with buffer D serving as NIR.
(LP)-ELISA The test was performed at the World Reference Laboratory for FMD as described by Hamblin et al. (1986a). A cut off threshold was used and samples with titres of 1.6 log~o or lower were considered negative.
Evaluation of tests Specificity, sensitivity and precision was determined according to Martin (1977 ); linear correlation of two variables, tests for correlation coefficients and variance analysis were as described by Croxton (1959). RESULTS
Initial tests on the influence of incubation time and temperature gave similar results as obtained previously (Sorensen and Lei, 1986) and incubation overnight at room temperature ( 2 0 - 2 4 ° C ) was subsequently used throughout. Non-specific reactivity of the occasional serum detected by high OD readings was eliminated by adding 1% normal rabbit serum to the buffer (buffer D ).
Specificity of the blocking ELISA Screening tests were carried out on the 1497 serum samples collected at the CSC slaughterhouse, Chinhoyi. Any sample with ODp readings below 50 was heated for 30 minutes at 56°C and retested. Defining all the cattle as being "non-diseased animals" the specificity of the test was calculated at the ODp50, the ODp40 and the ODp30 level. The results are given in Table 1. It appears that the specificity was high for all three types at the ODp30 level, but dropped especially for SATI at the ODp40 and the ODp50 level. For SAT3 it was very high even at the ODp50 level. The serum samples from DK and U K gave ODp values higher than 50 in screening tests with all 3 types except for one UK sample which repeatedly gave ODp values between 30 and 35 in the SAT1 test. The mean ODp values of NBS using buffer D as NIR appear in Table 2.
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TABLE 1 Determination of the specificity of the blocking ELISA for SAT1, SAT2 and SAT3 at the ODp50, ODp40 and ODp30 level using 1497 sera from "non-diseased" Zimbabwean cattle Test result
Non-diseased animals SAT 1
No. positive No. negative Total Specificity (%)
SAT2
SAT3
ODp50
ODp40
ODp30
ODp50
ODp40
ODp30
ODp50
ODp40
ODp30
28 1469 1497 98.1
11 1486 1497 99.3
0 1497 1497 100
8 1489 1497 99.5
6 1491 1497 99.6
3 1494 1497 99.8
1 1496 1497 99.9
0 1497 1497 100
0 1497 1497 100
TABLE 2 Determination of non-specific inhibition of SAT1, SAT2 and SAT3 with NBS in the blocking ELISA
Mean ODp S.D. No. of tests
SAT 1
SAT2
SAT3
72 9 25
71 9 25
62 9 25
Buffer D served as NIR.
Non-specific inhibition was demonstrated showing the importance of using NBS as NIR in screening tests.
Comparison of the blocking ELISA and the LP-ELISA Table 3 shows the results of testing 157 trivalently vaccinated cattle using the blocking ELISA and the LP-ELISA. It appears that the blocking ELISA picked up more positives of SAT 1 and SAT2 and in particular of SAT 1 even at the ODp30 level than the LP-ELISA. It gave more positives of SAT3 at the ODp50 level, the same number at the ODp40 level and fewer at the ODp30 level. One sample which was positive in the LP-ELISA for SAT 1 while negative in the blocking ELISA at the ODp30 level was positive at the ODp40 level. Two samples positive for SAT2 (titres 1.7 and 1.8 logl0) and 9 samples positive for SAT3 (titre range 1.7 to 2.1 logl0) in the LP-ELISA remained negative in the blocking ELISA at the ODp50 level. Defining all the vaccinated cattle as being "diseased animals" the sensitivity of the blocking ELISA at the ODp50, the ODp40 and the ODp30 level and of the LP-ELISA were calculated and given in Table 4. The sensitivity of the blocking ELISA was higher for SAT 1 and SAT2 and in particular for SAT1 even at the ODp30 level than that of the LP-ELISA. For SAT3 it was higher at the ODp50 level,
BLOCKING ELISAFOR DETECTION OF ANTIBODIESTO FMDV
259
TABLE 3 The number of positive and negative animals in the screening of 157 trivalently vaccinated cattle for antibodies against SAT1, SAT2, and SAT3 with the blocking ELISA and the LP-ELISA Blocking ELISA SAT 1
SAT2
SAT3
ODp50 ODp40 ODp30 OD50p
ODp40
OD30p
ODp50
ODp40
ODp30
p.
n.
p.
n.
p.
p.
p.
n.
p.
2 12
125 15
2 15
123 4 8 22
108 18
19 82 45 12 11 19
n.
LP-ELISA p. 89 0 n. 66 2
p.
n.
p.
n
p.
89 0 61 7
88 57
1 125 11 18
n.
n.
118 9 23 7
n.
p. = positive; n. = negative. TABLE 4 Determination of the sensitivity of the blocking ELISA at the ODp50, ODp40 and ODp30 level and of the LP-ELISA for SAT1, SAT2 and SAT3 using sera from 157 trivalently vaccinated "diseased" cattle Test result
Diseased animals Blocking ELISA
LP-ELISA
ODp50
ODp40
ODp30
Number positive Number negative Total number Sensitivity (%)
155 2 157 98.7
150 7 157 95.6
145 12 157 92.4
89 68 157 56.7
Number positive Number negative Total number Sensitivity (%)
143 14 157 91.1
140 17 157 89.2
131 26 157 83.4
127 30 157 80.9
Number positive Number negative Total number Sensitivity (%)
141 16 157 89.8
126 31 157 80.3
93 64 157 59.2
127 30 157 80.9
SAT1
SAT2
SAT3
similar at the ODp40 level and lower at the ODp30 level than that of the LPELISA. The sera of the cattle positive in the blocking ELISA at the ODp40 level were titrated. Titres from 0.9 1Oglo obtained in the LP-ELISA were recorded
260
K J
S()RENSEN
ET
~1.
irrespective of the cut off threshold of 1.6 log]o and the results are shown in Fig. 1. The correlation was highly significant for SAT1 and SAT2 and although the correlation coefficient was lower for SAT3 it was still significant at the 0.001 level. The cut off threshold of the LP-ELISA at 1.6 logm is indicated and the regression equation lines are shown. The slope for SAT1 and SAT2 was 1.0 and the average ratio between titres of the LP-ELISA and the blocking ELISA was about 1:2.5 for SAT1 and about 1:2.2 for SAT2. For SAT3 the slope was 0.4 which gave higher titres in the high titre range and lower titres in the low titre range with the blocking ELISA than with the LPELISA. LP-ELISA LOG10 TITRE 3
2.5
SAT1
f ~
"
2 1.6
1.5
1
0.5 0.5
r
i
i
1 1.5 2.5 3 BLOCKING ELISA LOG10 TITRE
i
3.5
LP-ELISA LOG10 TITRE
LP-ELISA LOG10 TITRE
3
SAT3
2.5 i ;
2
2
1.6
1.6
1.5
1.5
1
1
0.5 0.5
1 1.5 2 2.5 3 BLOCKING ELISA LOG10 TITRE
3.5
0.5 0.5
]
i
i
P
r
1 1.5 2 2.5 3 BLOCKING ELISA LOGIO TITRE
i
3.5
Fig. 1. Comparison of the blocking ELISA titres with the LP-ELISA titres using sera from trivalently vaccinated cattle. The correlation coefficients were: S A T l = 0 . 7 8 ; SAT2=0.88; SAT3 = 0.33. Bar-lines indicate the standard deviations. A total of 145, 140 and 127 sera were included for SAT 1, SAT2 and SAT3, respectively.
BLOCKING
ELISA
FOR
DETECTION
OF ANTIBODIES
TO
261
FMDV
TABLE5 The correlation coefficients, intercepts and slopes of the regression equation lines for SAT1, SAT2 and SAT3 calculated from the comparison of the ODp values of a single dilution of sera from trivalently vaccinated cattle with the corresponding log~o end point titres
No. of samples Correlation coefficient Y axis Intercept (a) Slope (b)
SAT1
SAT2
SAT3
117 - 0.97
90 - 0.95
97 - 0.95
2.8 - 0.018
3.2 - 0.025
3.1 - 0.023
X - a x i s = O D p values of serum diluted 1:80. Y-axis = log~o end point titres. END POINT LOG10 TITRE
2.5
2
1.5
1
0.5 0.5
i
i
i
i
P
1 1.5 2 2.5 3 SINGLE DILUTION LOG10 TITRE
i
3.5
END POINT LOGIO TITRE
END POINT LOG10 TITRE 3
3
2.5
2.5
2
1.5
1.5
1
1
0.5 0.5
r
i
i
i
1 1.5 2 2.5 3 SINGLE DILUTION LOGIO TITRE
t
3.5
0.5 0.5
i
i
i
i
i
1 1.5 2 2.5 3 SINGLE DILUTION LOGIO TITRE
i
3.5
Fig. 2. C o m p a r i s o n o f the end point titres with the single dilution titres o b t a i n e d with the blocking ELISA using r a n d o m l y selected samples s u b m i t t e d for F M D testing over a 3 - m o n t h period. The correlation coefficients were: SAT1 = 0.95; SAT2-- 0.97; SAT3 = 0.95. Bar-lines indicate the s t a n d a r d deviations. A total o f 72, 77 a n d 95 sera were included for SATI, SAT2 a n d SAT3, respectively.
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K.J. SORENSEN ET AI_.
TABLE 6 Examination of the precision of the blocking ELISA by titrating 3 sera 4 times against SAT l, SAT2 and SAT3 (rows) on each of 4 different days (columns) Testno.
BlockingELISA SAT1
SAT2
SAT3
1
2
3
4
1
2
3
4
1
2
3
4
1 2 3 4
2.3 2.2 2.2 2.3
2.2 2.2 2.2 2.3
2.0 2.2 2.0 2.0
1.9 2.0 2.0 2.1
2.3 2.3 2.2 2.3
2.2 2.5 2.4 2.3
2.0 2.2 2.3 2.1
2.2 2.4 2.4 2.3
2.6 2.4 2.2 2.3
2.4 2.5 2.5 2.5
2.3 2.5 2.4 2.4
2.2 2.4 2.4 2.3
Mean S.D.
2.25 0.06
2.23 0.05
2.05 0.10
2.00 0.08
2.28 0.05
2.35 0.13
2.15 0.13
2.33 0.10
2.38 0.17
2.48 0.05
2.40 0.08
2.33 0.10
Single dilution titres obtained with the blocking ELISA It was found that the maximum titre range was covered using a serum dilution of 1 : 80 which was applied in subsequent tests. The ODp values calculated on serum diluted 1 : 80 and the corresponding blocking ELISA loglo end point titres for SAT1, SAT2 and SAT3 were compared using sera of the trivalently vaccinated animals. The correlation coefficients, intercepts and slopes of the regression equation lines are given in Table 5. For calculation of single dilution titres the estimating equation Y = a + bX was used, where Y was the loglo single dilution titre to be calculated and X the known ODp value. To establish the correlation between the single dilution titres and the end point titres random samples from 23 submissions to the laboratory over a 3 month period were used. The estimating equations: Y = 2 . 8 - 0 . 0 1 8 X , Y = 3 . 2 + 0 . 0 2 5 X and Y = 3 . 1 - 0 . 0 2 3 X for SAT1, SAT2 and SAT3 respectively, were derived from the data in Table 5 and were used for calculation of the single dilution titres. The results are shown in Fig. 2. It appears that there was a positive linear correlation between the titres obtained with the 2 methods. A deviation was observed in the low titre range up to 1.5 loglo especially for SAT 1 and SAT3.
Precision of the blocking ELISA Three sera with antibodies against SAT1, SAT2 and SAT3 were titrated against the 3 types using the single dilution method. Four titres were obtained in each test run on each of 4 different days. The results are given in Table 6. The difference between column means i.e. titres obtained on different days was not significant for SAT3 ( P > 0 . 1 ) . It was slightly higher for SAT2 (0.1 > P > 0.05 ) and highest for SAT 1 (0.01 > P > 0.001 ). Adjustment factors were calculated for each day by dividing the known mean titre of a control
BLOCKING ELISA FOR DETECTION OF ANTIBODIES TO FMDV
263
serum with the titre obtained with the same control serum on the particular day. By adjusting the SAT 1 results the difference between column means decreased ( 0 . 0 2 5 > P > 0 . 0 1 ) . Adjustment did not improve the SAT2 and the SAT3 results. DISCUSSION
This work was prompted by the need for a test to be used in FMD control which united high output and low cost with high sensitivity, high specificity and precision. Blocking ELISA had very favourable logistics and low cost compared to the LP-ELISA. Thus 80 samples were examined per plate with the blocking ELISA, while the LP-ELISA incorporated only 24 samples per plate and required two different types of plate for each test. In addition the use of additives to buffers such as NBS and normal rabbit serum was low compared with the LP-ELISA. The application of the biotin-avidin system made it possible to use the same i m m u n e rabbit serum for the capture IgG and for the biotinylated IgG suspensions. The purification and biotinylation of IgG was uncomplicated and performed in 2 to 3 days. Although the procedure resulted in a 1 : 10 dilution of the i m m u n e sera, the biotinylated IgG suspensions were used in dilutions of 1 : 1500 to 1 : 3000. When reading the SAT 1 and SAT2 tests at the ODp30 level and the SAT3 test at the ODp40 level the sensitivity was high compared to the LP-ELISA. Adopting a zone for doubtful results between the ODp30 and ODp40 levels for SAT 1 and SAT2; and between the ODp40 and ODp50 levels for SAT3 may further add to the sensitivity. At the same time the specificity was high. However differences were found between the FMD virus types. While the high specificity obtained for SAT 1 at the ODp30 level dropped at the ODP40 and ODP50 levels a high specificity was obtained for SAT3 at all 3 levels. The specificity for SAT2 may be higher than stated because the 3 samples with ODp values lower than 30 (Table 1 ) may have originated from genuinely positive animals involved in the SAT2 outbreak in 1989. The striking difference in sensitivity between the two tests may be explained by the finding, as it appears in Fig. 1, that many animals were positive in the blocking ELISA but gave titres below the cut off threshold of 1.6 loglo in the LP-ELISA and were therefore considered negative in that test. It was also notable that the blocking ELISA gave higher titres than the LP-ELISA in both the low and the high titre range for SAT 1 and SAT2 with a slope of the regression equation line of 1.0. For SAT3 the slope was lower and the blocking ELISA gave higher titres in the high titre range and lower titres in the low titre range than the LP-ELISA. The threshold of the LP-ELISA was introduced for the sake of achieving specificity (Hamblin et al., 1986b). Nevertheless, the Department of Veteri-
264
K.J. SORENSEN ET AL.
nary Services in Zimbabwe had experienced problems with false positive results with different types, including European ones which have never occurred in the country, but notably with SAT3 (Hargreaves, pers. commun. ). The threshold was established for all seven serotypes from the results obtained by the testing of about 300 British FMD free cattle (Hamblin et al. 1986b) and it may not apply to tests on Zimbabwean cattle. Buffer served as non-inhibited reference (NIR) in the LP-ELISA where normal bovine serum (NBS) was used in the blocking ELISA and the observed non-specific inhibition especially of SAT3 with NBS (Table 2 ) concurred with the finding that a number of samples were found positive in the LP-ELISA while negative in the blocking ELISA against SAT3 (Table 3). Hamblin et al. (1986a) observed cross reaction when testing type specific SAT1 positive serum in the SAT3 test with the LP-ELISA. Hamblin et al. (1986b) also observed a poor correlation between the LP-ELISA and the virus neutralisation test when testing sera from animals which had been vaccinated against more than one FMD virus type. The present results with SAT3 on sera from trivalently vaccinated cattle may call for further examination, including the virus neutralisation test, which can be persued in later work. The correlation between the ODp values obtained with sera diluted 1:80 and the corresponding end point titres in the blocking ELISA was very high for all 3 types. This was compatible with results obtained by Westbury et al. ( 1988 ). It allowed the calculation of single dilution titres using the estimating equations. The titre determination on a single dilution of serum was extremely resources saving compared to end point titrations and proved to be a reliable and a precise method for the quantification of antibodies in sera found positive in the screening test. It may also be useful for the assessment of antibody levels of large populations, for example the cattle in the vaccinated zones of Zimbabwe. However, this has yet to be shown. ACKNOWLEDGEMENTS
This study was supported by grants from the Danish International Development Agency and the British Overseas Development Administration. We thank Dr. B. Guilloteau, Botswana Vaccine Institute for support and for supplying the crude and purified antigens. We thank Dr. P. Kitching, the World Reference Laboratory for FMD for fruitful discussions and for carrying out the LP-ELISA tests.
REFERENCES Condy, J.B., 1979. A history of foot and mouth disease in Rhodesia. Rhod. Vet. J. 10: 2-10. Croxton, F.E., 1959. In: F.E. Croxton (Editor), Elementary Statistics with Applications in
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Medicine and Biological Sciences. Dover Publications, Inc., New York, NY, 109-143 and 288-318. Fayet, M.T., Roumiantzeff, M. and Fargeaud, D., 1969. Analysis by zonal sucrose gradient centrifugation of virus particles contained in a foot and mouth disease virus preparation. Rev. Immunol., 33: 335-344. Guinet, J.J., Falconer, J., Dupasquier, M. and Mannathoko, M., 1982. Report on two years' experience in foot and mouth disease vaccine production at the Botswana Vaccine Institute. Rev. Sci. Tech. Off. Int. Epiz., 1: 337-348. Hamblin, C., Barnett, I.T.R. and Hedger, R.S., 1986a. A new enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against foot-and-mouth disease virus I. Development and method of ELISA. J. Immunol. Methods, 93:115-121. Hamblin, C., Barnett, I.T.R. and Crowther, J.R., 1986b. A new enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against foot-and-mouth disease virus II. Application. J. Immunol. Methods, 93: 123-129. Hargreaves, S.K., Director of Veterinary Services, Zimbabwe, personal communication. Have, P. and Holm Jensen, M., 1983. Detection of antibodies to FMDV type O by enzymelinked immunosorbent assay (ELISA). Report of the session of the research group of the standing technical committee of the European Commission for the control of Foot-and-Mouth Disease. Lelystad, The Netherlands, 20-22 September, 44-51. Hof Jorgensen, R., 1989. An international comparison of different laboratory tests for the diagnosis of bovine leukosis: Suggestions for international standardization. Vet. Immunol. Immunopathol., 22: 293-297. Martin, S.W., 1977. The evaluation of tests. Can. J. Comp. Med., 41: 19-25. Preaud, J.M., Dubourget, P.H., Detraz, N. and Lombard, M., 1989. Inactivation of foot and mouth disease viruses by ethylene imine kinetics and process control. International Symposium on Foot and Mouth Disease, Ankara, 6-8 June 1989. Sorensen, K.J. and Lei, J.C., 1986. Aujeszky's disease: Blocking ELISA for the detection of serum antibodies. J. Virol. Methods, 13:171-181. Sorensen, K.J., Ammendrup, S., Bitsch, V., Kirkegaard Petersen, B. and Warming, M., 1986. Aujeszky's disease: Blocking ELISA for large scale screening of serum antibodies. Proc. Int. Pig Vet. Soc. Congr. (Barcelona), 347. Westbury, H.A., Pornchai Chamnanpood, Suchinta Tangchaitrong and Doughty, W.J., 1988. Single dilution ELISA for detection of serum antibody to foot-and-mouth disease virus in cattle. Vet. Microbiol., 18: 273-283.
253
Foot-and-mouth disease: detection of antibodies in cattle sera by blocking ELISA K.J. Sorensen, R.L. Madekurozwa and P. Dawe Veterinary Research Laboratory, Harare, Zimbabwe (Accepted 13 February 1992)
ABSTRACT Sorensen, K.J., Madekurozwa, R.L. and Dawe, P., 1992. Foot-and-mouth disease: detection of antibodies in cattle sera by blocking ELISA. Vet. Microbiol., 32: 253-265.
A blocking ELISA was developed for the detection of antibodies to foot-and-mouth disease virus SAT1, SAT2 and SAT3 and for the quantification of antibodies on a single dilution of serum. The avidin-biotin system was used. The test was compared with the liquid-phase ELISA executed at the World Reference Laboratory for foot-and-mouth disease. It was found to have favourable logistics and combined high specificity with high sensitivity. The quantitative test using a single dilution of serum was resource saving and proved to be a reliable and precise method for the assessment of antibody levels.
INTRODUCTION
The control of foot-and-mouth disease ( F M D ) is of major importance to Zimbabwe in particular because of exports of animals and animal produce. As a result o f the economic implications of the disease major resources are invested in its control. The confinement of the buffalo herds to the national parks and hunting areas and their segregation from the domestic cattle is sustained by hundreds of kilometers of game fences. Vaccinations of cattle herds are carried out twice a year in zones immediately adjacent to the national parks. Buffer zones are maintained between the vaccination zones and the FMD free area. The division into zones is sustained by thousands of kilometers of cattle fences and cattle movement between and out of the zones is controlled. Large scale serological examinations are needed to ascertain the freedom from infection or vaccination of the cattle in the buffer zones and in the FMD free area and also to assess antibody levels in the vaccinated cattle. It therefore became necessary to introduce an ELISA which would give high Correspondence to: K.J. Sorensen, Veterinary Research Laboratory, P.O. Box 8101, Causeway, Harare, Zimbabwe.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
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sensitivity, high specificity and precision. As the veterinary Research Laboratory (VRL) in Harare had no security facilities for handling live FMD virus it also had to be a safe test. In addition the concern for logistics and for keeping expenditure as low as possible required it to be simple and give a high output. Liquid phase (LP)-ELISA was found to be sensitive ( Hamblin et al., 1986a ) but the Department of Veterinary Services in Zimbabwe had experienced problems with the specificity of the test (Hargreaves, pers. c o m m u n . ) . The cost of the test is high because it requires serial dilution of the samples and for the use of 2 different types of plates for each test. Blocking ELISA has previously been applied to FMD control (Have and Holm Jensen, 1983; Westbury et al., 1988). It has been used with great efficiency in the control and eradication of other diseases such as Aujeszky's disease (Sorensen and Lei, 1986; Sorensen et al., 1986) and enzootic bovine leucosis (Hof Jorgensen, 1989) and has proved to unite precision, high sensitivity and specificity with relative simplicity and low cost. Therefore a solid phase blocking ELISA was developed using inactivated virus produced at Botswana Vaccine Institute (BVI), Gaborone. This paper describes the test and presents comparative results with the LP-ELISA. MATERIALS AND METHODS
Antigen Crude inactivated FMD virus type SAT 1 BOT1/77, SAT 2 ZIM 7/89 and SAT 3 ZIM 9/81 was used as antigen. The virus was grown on bovine tongue epithelium using the Frenkel technique according to standard procedure at BVI (Guinet et al., 1982 ). It was inactivated with ethylene imine after clarification and filtration (Preaud et al., 1989). Purified inactivated 146 S particles obtained by sucrose gradient centrifugation (Fayet et al., 1969) was used for immunization of rabbits. Immune sera Two rabbits were used for each of the three SAT types. Each rabbit received two inoculations three weeks apart with about 25 #g of 146 S particles emulsified in equal volumes of Freund's complete adjuvant. The rabbits were bled repeatedly from ear veins three to five weeks after the last injection. The collected serum fractions were pooled, sodium azide was added to 0.1% and they were stored at 4 ° C. Isolation of rabbit IgG DEAE Affi-Gel Blue agarose (Bio-Rad, cat. 153-7307) was used to isolate the IgG fraction. The procedure recommended by the producer was applied. Briefly: 1 ml of serum was dialysed overnight at 4°C against 0.02 M KzHPO4,
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255
pH 8.0, 0.02% NAN3. The dialysed serum was layered on top o f a 5 ml econocolumn (Bio-Rad) which had been filled with DEAE Affi-Gel Blue agarose and prewashed with 2 bed volumes of 0.02 M K2HPO4, pH 8.0, 0.2% NAN3. The IgG was eluted with 2 bed volumes of the latter buffer and all the eluate ( 10 ml) was collected. The resultant IgG suspension was stored at 4 oC.
Biotinylation of rabbit IgG Biotinamidocaproate N-hydroxysuccinimide ester (SIGMA, cat. B2643 ) dissolved in dimethylsulfoxide was used for biotinylation. Initially the IgG solution was dialysed overnight at 4°C against O. 1 M NaHCO3 pH 8.5. The protein concentration was determined by spectrophotometry at a wavelength at 280 n m using an extinction coefficient of 1.4 for IgG. One part of biotinamidocaproate N-hydroxysuccinimide ester was added to eight parts of protein and incubated for 4 hours at room temperature. The solution was dialysed overnight at 4°C against PBS pH 7.2. To the biotinylated IgG suspension was added sodium azide to a concentration of 0.1% and normal bovine serum to a concentration of 20%. It was stored at 4 oC.
Test samples A total of 1497 blood samples from herds within the FMD free area in Mashonaland West province of Zimbabwe (from which the EEC imports beef) were collected over a period of seven months during 1990 at the Cold Storage Commission (CSC) slaughterhouse, Chinhoyi where non-vaccinated and non-diseased cattle were slaughtered. An outbreak of FMD SAT2 had occurred on one farm in the area in 1989. The farm was depopulated and a ring vaccination zone was established. Two outbreaks of F M D SAT2 were recorded in the late seventies but except for that the province had been free of FMD and vaccinations had not been carried out. Outbreaks with SAT 1 and SAT3 have never occurred (Condy, 1979). Serum samples ( n = 2 8 0 ) from cattle in Denmark (DK) and England ( U K ) were kindly supplied from the State veterinary Institute for Virus Research, Lindholm, Denmark and from the Central veterinary Laboratory, Weybridge, England. The above mentioned sera were screened with the blocking ELISA and used for assessing the specificity of the test. Sera were collected from a total of 157 cattle in 3 herds vaccinated with trivalent SAT 1, SAT2 and SAT3 vaccine produced at BVI in a ring vaccination zone instituted in May 1989 on the occasion of an outbreak of FMD SAT2 in the Midlands province. The sera were used for comparative testing with the LP-ELISA and the blocking ELISA. They were also used to derive the correlation between the end point titres and the ODp values obtained on a single dilution of serum with the blocking ELISA. Randomly selected sera from 23 submissions sent to the laboratory for FMD
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K.J. SORENSEN ET A_l.
testing over a period of 3 months were used to test the correlation between end point titres and single dilution titres.
Normal bovine serum (NBS) Sera were collected from FMD free cattle at the CSC slaughterhouse, Chinhoyi mentioned above and pooled. All the sera were tested individually for antibodies against FMDV SAT 1, SAT2 and SAT3 with negative results before pooling, eliminating the chance of contaminating antibodies.
Blocking ELISA (a) Buffers and substrate: 1. Buffer A: 50 m M NaHCO3, 0.02% NaN3 pH 9.6 2. Buffer B: 15 m M Na2HPO4, 3 m M KH2PO4, 500 m M NaCI, 0.05% Tween 20 pH 7.2.3. Buffer C: buffer B containing 3% normal bovine serum. 4. Buffer D: 34 m M Na2HPO4, 4 m M KH2PO4, 140 m M NaC1, 0.05% Tween 20, 0.02% NAN3, 1% normal rabbit serum pH 7.5.5. Substrate: 0.1% tetramethyl-benzidine TMB (Aldrich Chemical Co. Ltd, cat. 86,033-6) dissolved in absolute ethanol and further diluted 1 : I0 in a buffer consisting of 24 mM citric acid, 51 m M Na2HPO4, 0.025% hydrogen peroxide (30% H202), pH 5.0. (b) Consumables and equipment: 1. Plates: Nunc Immunoplate Maxisorp cat. 4-42404.2. Conjugate: Peroxidase conjugated Avidin, Dakopatt, cat. p347. 3. Microplate washer: Titertec 120. 4. Microplate photometer: Titertek Multiscan. (c) Performance Q(the test: The test was carried out as previously described for Aujeszky's disease (Sorensen and Lei, 1986) with some modifications. Briefly: ( 1 ) Plates were coated overnight at 4°C with the highest dilution of rabbit IgG in buffer A giving m a x i m u m catchment of antigen. (2) Antigen was added at an appropriate dilution in buffer B to give an OD value of about 1.0 and incubated at 37°C for 1 hour. (3) Dilution of test samples in buffer D. One well was used per sample diluted 1 : 10 for screening. Serial dilutions for titre determinations were carried out with one well per dilution step. Incubation was overnight at room temperature (20-24 ° C ). Six wells with NBS, 2 wells with a weak positive and 2 wells with a strong positive control serum all diluted 1:10 in buffer D and 6 wells with buffer D only were included on each plate. The wells with NBS or buffer D served as non-inhibited references (NIR). (4) Biotinylated IgG suspension diluted in buffer C to give an OD value of about 1.0 was added and incubated at 37°C for 1 hour. (5) Conjugate diluted 1:10 000 in buffer C was added and incubated for 30 rain at 37 ° C. (6) Substrate was added and incubated at room temperature for about 10 min. (7) Sulphuric acid 1 M was added to stop the reaction. Hundred microliter volumes were used in all steps. Steps 1 to 5 were followed by a wash of 3 cycles without soak periods except for step 3 where the wash was stopped after the 1st cycle and step 5 where a soak period of 30 s
BLOCKING ELISA FOR DETECTION OF ANTIBODIES TO FMDV
257
was included on the 2nd cycle. Buffer B was used for washing. OD readings were carried out at a wavelength of 450 nm. ODp values were calculated using the formula: ODp = (sample OD X 100) / (mean OD NIR) Results of the screenings were read at the ODp30, ODp40 and at the ODp50 levels on samples diluted 1 : 10 with NBS serving as NIR. End point titres were expressed as the reciprocal value of the dilution giving an ODp value of 50 with buffer D serving as NIR. ODp values used for determination oftitres on a single dilution of serum (single dilution titre) were calculated with buffer D serving as NIR.
(LP)-ELISA The test was performed at the World Reference Laboratory for FMD as described by Hamblin et al. (1986a). A cut off threshold was used and samples with titres of 1.6 log~o or lower were considered negative.
Evaluation of tests Specificity, sensitivity and precision was determined according to Martin (1977 ); linear correlation of two variables, tests for correlation coefficients and variance analysis were as described by Croxton (1959). RESULTS
Initial tests on the influence of incubation time and temperature gave similar results as obtained previously (Sorensen and Lei, 1986) and incubation overnight at room temperature ( 2 0 - 2 4 ° C ) was subsequently used throughout. Non-specific reactivity of the occasional serum detected by high OD readings was eliminated by adding 1% normal rabbit serum to the buffer (buffer D ).
Specificity of the blocking ELISA Screening tests were carried out on the 1497 serum samples collected at the CSC slaughterhouse, Chinhoyi. Any sample with ODp readings below 50 was heated for 30 minutes at 56°C and retested. Defining all the cattle as being "non-diseased animals" the specificity of the test was calculated at the ODp50, the ODp40 and the ODp30 level. The results are given in Table 1. It appears that the specificity was high for all three types at the ODp30 level, but dropped especially for SATI at the ODp40 and the ODp50 level. For SAT3 it was very high even at the ODp50 level. The serum samples from DK and U K gave ODp values higher than 50 in screening tests with all 3 types except for one UK sample which repeatedly gave ODp values between 30 and 35 in the SAT1 test. The mean ODp values of NBS using buffer D as NIR appear in Table 2.
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TABLE 1 Determination of the specificity of the blocking ELISA for SAT1, SAT2 and SAT3 at the ODp50, ODp40 and ODp30 level using 1497 sera from "non-diseased" Zimbabwean cattle Test result
Non-diseased animals SAT 1
No. positive No. negative Total Specificity (%)
SAT2
SAT3
ODp50
ODp40
ODp30
ODp50
ODp40
ODp30
ODp50
ODp40
ODp30
28 1469 1497 98.1
11 1486 1497 99.3
0 1497 1497 100
8 1489 1497 99.5
6 1491 1497 99.6
3 1494 1497 99.8
1 1496 1497 99.9
0 1497 1497 100
0 1497 1497 100
TABLE 2 Determination of non-specific inhibition of SAT1, SAT2 and SAT3 with NBS in the blocking ELISA
Mean ODp S.D. No. of tests
SAT 1
SAT2
SAT3
72 9 25
71 9 25
62 9 25
Buffer D served as NIR.
Non-specific inhibition was demonstrated showing the importance of using NBS as NIR in screening tests.
Comparison of the blocking ELISA and the LP-ELISA Table 3 shows the results of testing 157 trivalently vaccinated cattle using the blocking ELISA and the LP-ELISA. It appears that the blocking ELISA picked up more positives of SAT 1 and SAT2 and in particular of SAT 1 even at the ODp30 level than the LP-ELISA. It gave more positives of SAT3 at the ODp50 level, the same number at the ODp40 level and fewer at the ODp30 level. One sample which was positive in the LP-ELISA for SAT 1 while negative in the blocking ELISA at the ODp30 level was positive at the ODp40 level. Two samples positive for SAT2 (titres 1.7 and 1.8 logl0) and 9 samples positive for SAT3 (titre range 1.7 to 2.1 logl0) in the LP-ELISA remained negative in the blocking ELISA at the ODp50 level. Defining all the vaccinated cattle as being "diseased animals" the sensitivity of the blocking ELISA at the ODp50, the ODp40 and the ODp30 level and of the LP-ELISA were calculated and given in Table 4. The sensitivity of the blocking ELISA was higher for SAT 1 and SAT2 and in particular for SAT1 even at the ODp30 level than that of the LP-ELISA. For SAT3 it was higher at the ODp50 level,
BLOCKING ELISAFOR DETECTION OF ANTIBODIESTO FMDV
259
TABLE 3 The number of positive and negative animals in the screening of 157 trivalently vaccinated cattle for antibodies against SAT1, SAT2, and SAT3 with the blocking ELISA and the LP-ELISA Blocking ELISA SAT 1
SAT2
SAT3
ODp50 ODp40 ODp30 OD50p
ODp40
OD30p
ODp50
ODp40
ODp30
p.
n.
p.
n.
p.
p.
p.
n.
p.
2 12
125 15
2 15
123 4 8 22
108 18
19 82 45 12 11 19
n.
LP-ELISA p. 89 0 n. 66 2
p.
n.
p.
n
p.
89 0 61 7
88 57
1 125 11 18
n.
n.
118 9 23 7
n.
p. = positive; n. = negative. TABLE 4 Determination of the sensitivity of the blocking ELISA at the ODp50, ODp40 and ODp30 level and of the LP-ELISA for SAT1, SAT2 and SAT3 using sera from 157 trivalently vaccinated "diseased" cattle Test result
Diseased animals Blocking ELISA
LP-ELISA
ODp50
ODp40
ODp30
Number positive Number negative Total number Sensitivity (%)
155 2 157 98.7
150 7 157 95.6
145 12 157 92.4
89 68 157 56.7
Number positive Number negative Total number Sensitivity (%)
143 14 157 91.1
140 17 157 89.2
131 26 157 83.4
127 30 157 80.9
Number positive Number negative Total number Sensitivity (%)
141 16 157 89.8
126 31 157 80.3
93 64 157 59.2
127 30 157 80.9
SAT1
SAT2
SAT3
similar at the ODp40 level and lower at the ODp30 level than that of the LPELISA. The sera of the cattle positive in the blocking ELISA at the ODp40 level were titrated. Titres from 0.9 1Oglo obtained in the LP-ELISA were recorded
260
K J
S()RENSEN
ET
~1.
irrespective of the cut off threshold of 1.6 log]o and the results are shown in Fig. 1. The correlation was highly significant for SAT1 and SAT2 and although the correlation coefficient was lower for SAT3 it was still significant at the 0.001 level. The cut off threshold of the LP-ELISA at 1.6 logm is indicated and the regression equation lines are shown. The slope for SAT1 and SAT2 was 1.0 and the average ratio between titres of the LP-ELISA and the blocking ELISA was about 1:2.5 for SAT1 and about 1:2.2 for SAT2. For SAT3 the slope was 0.4 which gave higher titres in the high titre range and lower titres in the low titre range with the blocking ELISA than with the LPELISA. LP-ELISA LOG10 TITRE 3
2.5
SAT1
f ~
"
2 1.6
1.5
1
0.5 0.5
r
i
i
1 1.5 2.5 3 BLOCKING ELISA LOG10 TITRE
i
3.5
LP-ELISA LOG10 TITRE
LP-ELISA LOG10 TITRE
3
SAT3
2.5 i ;
2
2
1.6
1.6
1.5
1.5
1
1
0.5 0.5
1 1.5 2 2.5 3 BLOCKING ELISA LOG10 TITRE
3.5
0.5 0.5
]
i
i
P
r
1 1.5 2 2.5 3 BLOCKING ELISA LOGIO TITRE
i
3.5
Fig. 1. Comparison of the blocking ELISA titres with the LP-ELISA titres using sera from trivalently vaccinated cattle. The correlation coefficients were: S A T l = 0 . 7 8 ; SAT2=0.88; SAT3 = 0.33. Bar-lines indicate the standard deviations. A total of 145, 140 and 127 sera were included for SAT 1, SAT2 and SAT3, respectively.
BLOCKING
ELISA
FOR
DETECTION
OF ANTIBODIES
TO
261
FMDV
TABLE5 The correlation coefficients, intercepts and slopes of the regression equation lines for SAT1, SAT2 and SAT3 calculated from the comparison of the ODp values of a single dilution of sera from trivalently vaccinated cattle with the corresponding log~o end point titres
No. of samples Correlation coefficient Y axis Intercept (a) Slope (b)
SAT1
SAT2
SAT3
117 - 0.97
90 - 0.95
97 - 0.95
2.8 - 0.018
3.2 - 0.025
3.1 - 0.023
X - a x i s = O D p values of serum diluted 1:80. Y-axis = log~o end point titres. END POINT LOG10 TITRE
2.5
2
1.5
1
0.5 0.5
i
i
i
i
P
1 1.5 2 2.5 3 SINGLE DILUTION LOG10 TITRE
i
3.5
END POINT LOGIO TITRE
END POINT LOG10 TITRE 3
3
2.5
2.5
2
1.5
1.5
1
1
0.5 0.5
r
i
i
i
1 1.5 2 2.5 3 SINGLE DILUTION LOGIO TITRE
t
3.5
0.5 0.5
i
i
i
i
i
1 1.5 2 2.5 3 SINGLE DILUTION LOGIO TITRE
i
3.5
Fig. 2. C o m p a r i s o n o f the end point titres with the single dilution titres o b t a i n e d with the blocking ELISA using r a n d o m l y selected samples s u b m i t t e d for F M D testing over a 3 - m o n t h period. The correlation coefficients were: SAT1 = 0.95; SAT2-- 0.97; SAT3 = 0.95. Bar-lines indicate the s t a n d a r d deviations. A total o f 72, 77 a n d 95 sera were included for SATI, SAT2 a n d SAT3, respectively.
262
K.J. SORENSEN ET AI_.
TABLE 6 Examination of the precision of the blocking ELISA by titrating 3 sera 4 times against SAT l, SAT2 and SAT3 (rows) on each of 4 different days (columns) Testno.
BlockingELISA SAT1
SAT2
SAT3
1
2
3
4
1
2
3
4
1
2
3
4
1 2 3 4
2.3 2.2 2.2 2.3
2.2 2.2 2.2 2.3
2.0 2.2 2.0 2.0
1.9 2.0 2.0 2.1
2.3 2.3 2.2 2.3
2.2 2.5 2.4 2.3
2.0 2.2 2.3 2.1
2.2 2.4 2.4 2.3
2.6 2.4 2.2 2.3
2.4 2.5 2.5 2.5
2.3 2.5 2.4 2.4
2.2 2.4 2.4 2.3
Mean S.D.
2.25 0.06
2.23 0.05
2.05 0.10
2.00 0.08
2.28 0.05
2.35 0.13
2.15 0.13
2.33 0.10
2.38 0.17
2.48 0.05
2.40 0.08
2.33 0.10
Single dilution titres obtained with the blocking ELISA It was found that the maximum titre range was covered using a serum dilution of 1 : 80 which was applied in subsequent tests. The ODp values calculated on serum diluted 1 : 80 and the corresponding blocking ELISA loglo end point titres for SAT1, SAT2 and SAT3 were compared using sera of the trivalently vaccinated animals. The correlation coefficients, intercepts and slopes of the regression equation lines are given in Table 5. For calculation of single dilution titres the estimating equation Y = a + bX was used, where Y was the loglo single dilution titre to be calculated and X the known ODp value. To establish the correlation between the single dilution titres and the end point titres random samples from 23 submissions to the laboratory over a 3 month period were used. The estimating equations: Y = 2 . 8 - 0 . 0 1 8 X , Y = 3 . 2 + 0 . 0 2 5 X and Y = 3 . 1 - 0 . 0 2 3 X for SAT1, SAT2 and SAT3 respectively, were derived from the data in Table 5 and were used for calculation of the single dilution titres. The results are shown in Fig. 2. It appears that there was a positive linear correlation between the titres obtained with the 2 methods. A deviation was observed in the low titre range up to 1.5 loglo especially for SAT 1 and SAT3.
Precision of the blocking ELISA Three sera with antibodies against SAT1, SAT2 and SAT3 were titrated against the 3 types using the single dilution method. Four titres were obtained in each test run on each of 4 different days. The results are given in Table 6. The difference between column means i.e. titres obtained on different days was not significant for SAT3 ( P > 0 . 1 ) . It was slightly higher for SAT2 (0.1 > P > 0.05 ) and highest for SAT 1 (0.01 > P > 0.001 ). Adjustment factors were calculated for each day by dividing the known mean titre of a control
BLOCKING ELISA FOR DETECTION OF ANTIBODIES TO FMDV
263
serum with the titre obtained with the same control serum on the particular day. By adjusting the SAT 1 results the difference between column means decreased ( 0 . 0 2 5 > P > 0 . 0 1 ) . Adjustment did not improve the SAT2 and the SAT3 results. DISCUSSION
This work was prompted by the need for a test to be used in FMD control which united high output and low cost with high sensitivity, high specificity and precision. Blocking ELISA had very favourable logistics and low cost compared to the LP-ELISA. Thus 80 samples were examined per plate with the blocking ELISA, while the LP-ELISA incorporated only 24 samples per plate and required two different types of plate for each test. In addition the use of additives to buffers such as NBS and normal rabbit serum was low compared with the LP-ELISA. The application of the biotin-avidin system made it possible to use the same i m m u n e rabbit serum for the capture IgG and for the biotinylated IgG suspensions. The purification and biotinylation of IgG was uncomplicated and performed in 2 to 3 days. Although the procedure resulted in a 1 : 10 dilution of the i m m u n e sera, the biotinylated IgG suspensions were used in dilutions of 1 : 1500 to 1 : 3000. When reading the SAT 1 and SAT2 tests at the ODp30 level and the SAT3 test at the ODp40 level the sensitivity was high compared to the LP-ELISA. Adopting a zone for doubtful results between the ODp30 and ODp40 levels for SAT 1 and SAT2; and between the ODp40 and ODp50 levels for SAT3 may further add to the sensitivity. At the same time the specificity was high. However differences were found between the FMD virus types. While the high specificity obtained for SAT 1 at the ODp30 level dropped at the ODP40 and ODP50 levels a high specificity was obtained for SAT3 at all 3 levels. The specificity for SAT2 may be higher than stated because the 3 samples with ODp values lower than 30 (Table 1 ) may have originated from genuinely positive animals involved in the SAT2 outbreak in 1989. The striking difference in sensitivity between the two tests may be explained by the finding, as it appears in Fig. 1, that many animals were positive in the blocking ELISA but gave titres below the cut off threshold of 1.6 loglo in the LP-ELISA and were therefore considered negative in that test. It was also notable that the blocking ELISA gave higher titres than the LP-ELISA in both the low and the high titre range for SAT 1 and SAT2 with a slope of the regression equation line of 1.0. For SAT3 the slope was lower and the blocking ELISA gave higher titres in the high titre range and lower titres in the low titre range than the LP-ELISA. The threshold of the LP-ELISA was introduced for the sake of achieving specificity (Hamblin et al., 1986b). Nevertheless, the Department of Veteri-
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K.J. SORENSEN ET AL.
nary Services in Zimbabwe had experienced problems with false positive results with different types, including European ones which have never occurred in the country, but notably with SAT3 (Hargreaves, pers. commun. ). The threshold was established for all seven serotypes from the results obtained by the testing of about 300 British FMD free cattle (Hamblin et al. 1986b) and it may not apply to tests on Zimbabwean cattle. Buffer served as non-inhibited reference (NIR) in the LP-ELISA where normal bovine serum (NBS) was used in the blocking ELISA and the observed non-specific inhibition especially of SAT3 with NBS (Table 2 ) concurred with the finding that a number of samples were found positive in the LP-ELISA while negative in the blocking ELISA against SAT3 (Table 3). Hamblin et al. (1986a) observed cross reaction when testing type specific SAT1 positive serum in the SAT3 test with the LP-ELISA. Hamblin et al. (1986b) also observed a poor correlation between the LP-ELISA and the virus neutralisation test when testing sera from animals which had been vaccinated against more than one FMD virus type. The present results with SAT3 on sera from trivalently vaccinated cattle may call for further examination, including the virus neutralisation test, which can be persued in later work. The correlation between the ODp values obtained with sera diluted 1:80 and the corresponding end point titres in the blocking ELISA was very high for all 3 types. This was compatible with results obtained by Westbury et al. ( 1988 ). It allowed the calculation of single dilution titres using the estimating equations. The titre determination on a single dilution of serum was extremely resources saving compared to end point titrations and proved to be a reliable and a precise method for the quantification of antibodies in sera found positive in the screening test. It may also be useful for the assessment of antibody levels of large populations, for example the cattle in the vaccinated zones of Zimbabwe. However, this has yet to be shown. ACKNOWLEDGEMENTS
This study was supported by grants from the Danish International Development Agency and the British Overseas Development Administration. We thank Dr. B. Guilloteau, Botswana Vaccine Institute for support and for supplying the crude and purified antigens. We thank Dr. P. Kitching, the World Reference Laboratory for FMD for fruitful discussions and for carrying out the LP-ELISA tests.
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