197 Appendix 24 A solid-phase competition ELISA for measuring antibody to foot-and-mouth disease virus N.P. Ferris a, A.N. Bulut b, T. Rendle a, F. Davidson a and D.K.J. Mackay c a b c Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, UK FMD Institute, PK 714, 06 044 Ulus, Ankara, Turkey Veterinary Medicines Directorate, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK Summary A solid-phase competition ELISA (SPCE) has been devised for the measurement of antibodies to foot-and-mouth disease (FMD) virus. The assay uses polyclonal antisera and inactivated purified 146S antigens of FMD virus and was compared with the liquid-phase blocking ELISA (LPBE) and virus neutralisation test (VNT) on a range of serum sets. The SPCE exhibited equal test sensitivity as the LPBE and VNT and a better specificity of reaction than the LPBE when using test sera at a 1:5 dilution and a cut-off point of 30 percentage inhibition of antigen. The assay thus retains the sensitivity and ease of use of the LPBE whilst being more robust and specific and offers an improvement for FMD virus antibody detection. Introduction The OIE prescribed tests for foot-and-mouth disease (FMD) serology are the virus neutralisation test (VNT; Golding et al., 1976) and the liquid-phase blocking ELISA (LPBE; Hamblin et al., 1986). The LPBE has since been adopted by a large number of laboratories worldwide (Mackay et al., 1994, 1998) to replace the VNT for routine screening because it is quicker, more reproducible, correlates well with the VNT and does not suffer from the biological variability that is inherent in the VNT. However, the test is not without its problems. The percentage of animals giving false-positive results varies according to the animal population particularly in animals which are stressed and can be as high as 4% (Haas, 1994). For this reason, it is usually recommended that low-positive LPBE results are confirmed by VNT, particularly for the purposes of international trade (Anon, 1996). The assay requires training and experience to yield reproducible results and is not particularly 'robust'. A major factor for the lack of robustness of the LPBE has been in the variable stability of inactivated antigens employed in the procedure. We decided that it would be worthwhile to examine a solid-phase competition ELISA (SPCE) for FMD virus serology using polyclonal antiserum reagents (as used by the LPBE)
198 and inactivated, purified 146S antigens of FMD virus which have been observed to be remain stable after long term (years) storage at 4 o C (N.P. Ferris, unpublished results) Materials and methodsmaterials and methodsmaterials and methodsmaterials and methods Viruses FMD virus types O 1 BFS 1860, A 22 IRQ 24/64 and C 1 Noville were grown on monolayers of BHK-21 cells, inactivated with 1 M binary ethyleneimine (BEI; Bahnemann, 1990), 146S antigens purified according to the method of Ferris et al., 1984 and stored at 4 o C for subsequent use in the SPCE. Test Serum Samples Four sets of positive sera were examined : i) reference sera obtained from cattle between 21 and 28 days after either infection or vaccination with single, defined strains of FMD virus of known origin; ii) sera collected sequentially following either infection (type O 1 BFS) or vaccination (types A 22 and C 1 ) with FMD virus; iii) sera generated for the FAO Collaborative Studies Phases XIII (Mackay et al., 1994) and XV (Mackay et al., 1998) and iv) 'problem' sera : sera from cattle or water buffaloes which had been found to be low positive by LPBE and/or VNT on serological testing of animals prior to international trade but in which there was no recorded history of previous vaccination or infection. Additionally, negative sera from the UK, Canada or elsewhere which originated from animals with no history of exposure to FMD virus and which had been tested by LPBE and/or VNT with negative results. Virus neutralisation test and Liquid-phase blocking ELISA (LPBE) Virus neutralisation tests (VNT) were performed in tissue culture grade microtitre plates using the method described by Golding et al. (1976). The LPBE was carried out as described by Hamblin et al. (1986). Solid-phase competition ELISA (SPCE) The final procedure adopted for the solid-phase competition ELISA (SPCE) was as follows (50 µl reagent volumes were used throughout, ELISA plates [Nunc Maxisorp immunoplates] incubated for 1 hour in 37 o C on a rotary shaker, unless otherwise stated, and plates washed three times with phosphate buffered saline [PBS, ph 7.4] containing 0.05% Tween 20 after each incubation step). Plates were coated with an optimal dilution of rabbit antiserum to FMD virus in carbonate/bicarbonate buffer, ph 9.6 and incubated overnight at +4 o C. Next an optimal dilution of 146S antigen of FMD virus homologous to
199 the rabbit antiserum was diluted in PBS containing 0.05% Tween 20 and phenol red indicator (PBST) and added to each well. Duplicate, two-fold dilutions of each test serum (from an initial dilution of 1:2.5) in PBST containing 10% normal serum of the species under test and 5% normal rabbit serum (blocking buffer 3) were performed in plates. Immediately homologous guinea pig antiserum, diluted to the optimal concentration in blocking buffer, was added to each well. After plate incubation, an optimal dilution of rabbit anti-guinea pig immunoglobulins conjugated to horse radish peroxidase in blocking buffer was added to each well. Substrate (0.05% H 2 O 2 )/chromogen (orthophenylene diamine) in citrate/phosphate buffer, ph 5.0 was next added. After 15 min incubation the reaction was stopped by adding 1.25 M sulphuric acid. The optical density (OD) of each well was read by using a spectrophotometer (Dynatech) with a 492 nm filter. Antibody titres were expressed as the last dilution of serum showing 30% inhibition of OD compared to the mean OD of the reaction control wells where serum was absent. Blocking Buffers Three different blocking buffers were evaluated using defined positive and negative sera: i) blocking buffer 1 - PBS with 0.05% Tween 20 containing 5% skimmed milk powder ("Marvel"); ii) blocking buffer 2 - PBS (ph 7.4) with 0.1% Tween 20 and containing 0.3% normal serum of the species under test and iii) blocking buffer 3 PBS (ph 7.4) with 0.05% Tween 20 and containing 10% normal serum of the species under test and 5% normal rabbit serum. Results Differentiation of positive from negative sera To evaluate the performance of the test, 57 positive reference sera for type O and 120 negative sera were titrated in the SPCE for antibody to FMD virus type O 1 BFS 1860 in a dilution series from 1:2.5 to 1:80. The results were analysed in terms of percentage inhibition of antigen at each serum dilution (Fig. 1). The dilution at which there was optimal differentiation of positive from negative sera was 1:5. All positive sera had more than 30% inhibition and all negative sera less than this value. At this dilution there was no overlap in the two populations using blocking buffer 3, in contrast to considerable overlap of the positive and negative populations using blocking buffers 2 and 3 (results not shown). Blocking buffer 3 was therefore used for all subsequent work. Having established a provisional cut-off for the test, large numbers of negative sera were then examined at a single dilution of 1:5 (753 sera against type O 1 and 216 against types A 22 and C, of which 113 sera were examined against all 3 serotypes). Frequency distributions showed the percentage inhibitions of the negative sera to be normally distributed (Fig. 2). For a normal population, establishing a cut-off at the mean + 3
200 standard deviations (SD's) should mean that less than 1% of the values exceed the cut-off. For the negative populations studied here, the mean % inhibitions for types O, A and C were 9, 10 and 10% respectively with standard deviations of 6% in each case. This gave cut-off values of 27, 28 and 28% for types O, A and C respectively. To simplify interpretation and to ensure a high level of specificity, the cut-off was therefore set at 30% for all serotypes. Using this cut-off, the specificity of the assays for types A and C was 100% (zero positive out of 216). For type O, the specificity was 99.3% as 5 out of 753 sera scored positive at the single dilution of 1:5. However, all 5 sera were negative in a titration ELISA (i.e. titre <1:5) raising the specificity of the overall testing procedure to 100% for this population. Correlation with LPBE End point titres were determined in the SPCE and LPBE for positive reference sera for each of the serotypes O, A and C (Fig. 3). Statistically significant correlations were measured for each serotype and all sera positive by LPBE were also positive by SPCE. This result also shows that the SPCE detects antibody to a wide variety of strains within each serotype. For each serotype a single strain of FMD virus was used as the antigen in ELISA, whereas a wide variety of strains from within each serotype was used for infection or vaccination of cattle to produce the reference sera. The dynamics of the antibody responses to infection (type O) and vaccination (types A and C) showed that antibody titres were similar irrespective of measurement by either the LPBE or SPCE (results not shown). Titres in the low positive/doubtful range were measured at 7 days post vaccination (dpv) and all animals were strongly seropositive by 14 dpv. The sensitivity and reproducibility of the SPCE were compared directly with the LPBE and the VNT. Five two-fold dilutions of a type O reference serum were prepared in normal negative bovine serum such that the estimated end point titre in the SPCE was "bracketed" in either direction. These sera were then randomly labelled from A to E and repeatedly examined blind by SPCE, LPBE and by VNT, together with the original positive reference serum (N; Table 1). The SPCE and the LPBE had the same sensitivity, both scoring the 1:160 dilution (B) positive and the 1:320 dilution (D) negative. The VNT was slightly less sensitive than either ELISA scoring the 1:160 dilution (B) doubtful (i.e. titre between 1:16 and 1:32) but the 1:80 dilution (E) positive (i.e. titre >1:45). In terms of reproducibility, both ELISA's generally had lower coefficients of variation (CV) than the VNT. The CV's of the sera in the SPCE were similar to but slightly higher than those in the LPBE. Sera from two FAO Standardisation Exercises were examined by SPCE, LPBE and the VNT. In the Phase XIII Exercise (Table 2), panels of four reference sera for each of the strains O 1 BFS 1860, A 22 IRQ 24/64 and C 1 Noville were created which varied in strength
201 from weak positive through to strong positive. Although the absolute values varied between assays, there was good correlation between the three assays. All the reference sera correctly scored positive in the SPCE with the exception of RS-4 for type O 1 BFS 1860 which was negative by half a dilution step (titre 1:3.5; cut-off 1:5). However, this serum was also doubtful by VNT with a titre of 1:32 (cut-off in VNT 1:45).There was also good correlation between the results of all three tests in examination of the Phase XV provisional reference sera (results not shown). Using as antigen either the homologous strain of FMD virus with which the animal was infected or a heterologous strain of the same or a different serotype, there was a high degree of cross-reactivity between the two strains of type O in each of the 3 assays but the extent of cross-reactivity was greatest in the SPCE (results not shown). Some degree of cross-reactivity between serotypes was observed in all three assays for all three serotypes. In all cases, there was less cross-reactivity between serotypes in the SPCE than in the LPBE or VNT, suggesting that the SPCE is more serotype specific than the other two tests. Of the 73 of 85 >doubtful sera= which were positive in the LPBE for antibody to type O, only 3 were positive in the SPCE, one of which was also positive by VNT (Table 3). Likewise for antibody to type A, only 4 out of 48 sera which were LPBE positive were also positive by SPCE and two of these were also VNT positive. Assuming that these sera were actually from FMD naive animals, which could not be proven beyond doubt, then the SPCE was considerably more specific than the LPBE for these 'doubtful' sera and was only slightly less specific than the definitive VNT. Discussion Analysing the results overall, higher concentrations of positive sera were required to achieve a given percentage inhibition in the SPCE than in the LPBE. This meant that the cut-off in the SPCE was at a lower dilution (1:5) than in the LPBE (1:40; Hamblin et al., 1986) and at a lower percentage inhibition (30% as opposed to 50%). Nevertheless, using this cut-off value, the sensitivities of the two ELISA's were almost identical (Table 1). Only some sera with the lowest positive titre by LPBE (1:45) just scored negative (1:3.5) in the SPCE. The SPCE was at least as sensitive (Table 1) and certainly more specific (Table 3) than the 'gold standard' VNT. Any new test for antibody to FMDV must score correctly reference sera which are recognised as international standards. The SPCE scored the provisional FAO reference sera for FMD virus types O 1 Manisa, A 22 IRQ 24/64 and C 1 Noville correctly (Table 2) and showed good correlation with both the LPBE and the VNT for panels of positive reference sera to FMD virus type O 1 BFS 1860, A 22 IRQ 24/64 and C 1 Noville. When using serology for diagnosis of FMD it is useful to have a test which is as broadly cross-reactive between strains of a given serotype, but as serotype-specific as possible. Examining selected
202 reference sera by SPCE, LPBE and VNT showed that the SPCE most closely matched these criteria. Although positive titres were recorded against serotypes other than the one used to infect the animal, their heterologous titres were universally low, and lower in comparison, than those recorded in the LPBE and the VNT. The ability of the SPCE to detect antibody generated by exposure to a wide variety of strains within a serotype is best shown in Fig 3. For all 3 serotypes, all reference sera collected between 21 and 28 days after either infection or vaccination with any strain within a serotype gave a positive reaction in the SPCE using as antigen purified FMD virus of the homologous serotype. This figure also shows a strong correlation between titres in the SPCE and in the LPBE (and the r values would have been higher if end point titres had been determined for all sera having an SPCE titre of greater than 1:160). The fact that some sera had a high titre by LPBE, but a low titre by SPCE and vice versa suggests that the population of antibodies measured in the two tests were not identical. This is to be expected as the LPBE measures both antibody which prevents binding of the antigen in the liquid phase to the immobilised rabbit antiserum and antibody which blocks the detecting guinea pig antiserum. In contrast, the SPCE relies only on competition between the test serum and the detecting guinea pig antiserum. The objective of the work reported here was to develop an ELISA for antibody to FMD virus which retained the sensitivity and ease of use of the LPBE whilst being more robust and, hopefully, more specific. A sensitive, specific and reliable SPCE has been developed to measure antibody to FMD virus to fulfill the objective. Work is now in hand to adopt the assay for use with the other FMD virus serotypes and to evaluate the test in day-to-day use in the WRL for FMD. A full presentation of this work is in preparation for submission to the Journal of Virological Methods. Acknowledgements This work was supported financially by the UK Ministry of Agriculture, Fisheries and Food (project number SE 1113). The authors wish to thank members of the International Vaccine Bank, Pirbright Laboratory and Dr J. Salt for provision of test sera. ReferencesReferencesReferencesReferences Anon. (1996). OIE Manual of Standards for Diagnostic Tests and Vaccines. Lists A and B diseases of mammals, birds and bees. Chapter 2.1.1. Foot and Mouth Disease, pp. 47-56. Bahnemann, H.G. (1990). Vaccine 8, 299-303. Ferris, N.P., Donaldson, A.I., Barnett, I.T.R. and Osborne, R.W. (1984). Revue Scientific et Technique de l'office International des Epizooties 3, 339-350. Golding, S.M., Hedger, R.S. and Talbot, P. (1976). Research in Veterinary Science 20, 142-
203 147. Haas, B. (1994). 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, Vienna, 19-22 September, 1994, 124-127. Hamblin, C., Barnett, I.T.R. and Hedger, R.S. (1986). Journal of Immunological Methods 93, 115-121. Mackay, D.K.J., Rendle, T. and Armstrong, R.M. (1994). 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, Vienna, 19-22 September, 1994, 128-145. Mackay, D.K.J., Rendle, T., and Kitching, R.P. (1998). 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, Aldershot, UK, 14-18 September, 1998, 148-157. Mackay, D.K.J., Bulut, A.N., Rendle, T., Davidson, F. and Ferris, N.P. In preparation for submission to the Journal of Virological Methods. Table 1 Two-fold dilutions (shown in brackets) of a positive serum (N) diluted in normal bovine serum and repeatedly tested by SPCE, LPBE and VNT. Test Sera C (1280) A (640) D (320) B (160) E (80) N (neat, undiluted serum) SPCE a b 0% c 0% 0.47 0.11 23% 0.80 0.08 10% 1.07 0.08 7% 2.50 0.15 6% LPBE 1.34 0% 1.34 0% 1.59 0.09 6% 1.80 0% 2.17 0.09 4% 3.29 0.17 5% a b c VNT 0.83 0.13 15% 0.92 0.21 23% 1.18 0.09 7.6% 1.39 0.32 23% 1.72 0.24 14% 3.13 0.09 3% Mean titre (n=5 for SPCE and LPBE; n=3 for VNT) of each serum as logarithmic value Standard deviation (SD) Co-efficient of variation Number in parenthesis shows the serum dilution
204 Table 2 Examination of the provisional reference sera from the FAO Phase XIII Standardisation Exercise by SPCE, LPBE and VNT Phase XIII O 1 BFS 1860 RS-1 RS-2 RS-3 RS-4 SPCE LPBE VNT 452 a 2048 912 2.66 b 3.31 2.96 80 1024 468 1.90 3.01 2.67 14 90 51 1.15 1.95 1.71 3.5 45 32 0.54 1.65 1.50 Phase XIII A 22 IRQ 24/64 RS-1 RS-2 RS-3 RS-4 SPCE LPBE VNT 113 1448 2.05 3.16 80 724 513 1.9 2.86 2.71 57 362 123 1.76 2.56 2.09 20 64 42 1.30 2.26 1.63 Phase XIII C 1 Noville RS-1 RS-2 RS-3 RS-4 a b SPCE LPBE VNT 905 2048 1230 2.96 3.31 3.09 160 1024 224 Left hand columns : arithmetic titre Right hand columns : logarithmic titre 2.20 3.01 2.35 57 362 78 1.76 2.56 1.89 7 45 32 0.85 1.65 1.51 Table 3 A panel of 85 >problem= sera were selected for examination by SPCE, LPBE and VNT on the basis of previous reactivity in the LPBE for import/export testing and assumed to originate from FMD virus naive cattle FMD virus Test result Test SPCE LPBE VNT O 1 BFS 1860 Number positive Number negative Specificity (%) 3 82 96.4 73 12 14.1 1 84 98.8 A 22 IRQ 24/64 Number positive Number negative Specificity (%) 4 81 95.2 48 37 43.5 2 83 97.6
205