FAO Collaborative Study Phase XVII: Standardisation of FMD Antibody Detection

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1 Appendix 28 FAO Collaborative Study Phase XVII: Standardisation of FMD Antibody Detection D J Paton, R M Armstrong, L S Turner, P A Hamblin, M Corteyn, D Gibson, J Anderson Institute for Animal Health, Ash Road, Pirbright, Woking Surrey GU24 0NF Introduction The aims of the current study were agreed at the FAO EU FMD meeting in Borovets, The previously agreed reference sera to O Manisa, A22 and C Noville were to be adopted by O.I.E. as official standards. New reference sera were to be prepared against O SKR, A Iran 96 and Asia 1 Shamir. These should be distributed for evaluation, along with a panel of coded proficiency test sera. New primary sera were prepared at the start of 2001, but the FMD epidemic severely delayed subsequent progress. During 2002, the candidate reference sera were distributed and this report is mainly concerned with their evaluation. No proficiency testing with a panel of coded sera was performed. Materials and Methods Candidate reference sera for each of the FMD strains O SKR, A Iran 96 and Asia 1 Shamir were derived by diluting strong positive original sera in a standard, pooled negative serum (Table 1). Strong positive original sera were obtained from cattle after either intradermolingual inoculation (O SKR) or vaccination, including a 21 day boost (A Iran 96 and Asia 1 Shamir). Participants (Table 2) were requested to examine the sera by both ELISA and virus neutralisation test (VNT). In the case of ELISA, participants were asked to use both the liquid phase blocking ELISA (LPBE) and the solid phase competition ELISA (SPCE) as well as other available tests, including those for non-structural (NS) antibodies. All laboratories received a protocol for a version of the SPCE modified from that of Mackay et al. (2001), along with the necessary reagents for a serotype O version of the test. Results Results were available from all but one of the laboratories that had been sent samples and these are summarized in Tables 3-6. For the standard tests, the cut-offs used at Pirbright have been applied for interpretation of the results, although it is appreciated that other interpretations may be in use. Some results are not tabulated, since they were not directly comparable due to differences in the way the tests were performed or in which the results were expressed. The negative serum was found negative in all tests, in all labs. 226

2 The serotype O and A VNT results were strain dependent and use of a non-homologous subtype of virus, such as O Manisa instead of O SKR, or A22 instead of A Iran 96, generally reduced the antibody titre (Table 3). For the serotype O sera, the strong positive, weak positive and cut-off sera had mean arithmetic titres of 1:193, 1:37 and 1:15 respectively from the three laboratories who tested it using the homologous virus. Equivalent mean homologous titres were 1:661, 1:148 and 1:57 for the serotype A and 1:88, 1:22 and 1:8 for the serotype Asia 1 sera respectively. The results of LPBE testing are summarised in Table 4, except for those of lab 2, which reported results for serotypes O and A as percentage inhibitions at a single dilution. In this case, the strong positive sera were scored positive, as was the weak positive O SKR serum. The weak positive A Iran 96 serum and both cut-off sera were scored negative in lab 2. For the other LPBE results, all but one laboratory had the correct result with each of the strong positive sera, whereas the weak positive and cut-off sera gave inconsistent qualitative results (Table 4). There were also wide fluctuations in the quantitative results. For example, two labs found all of the type A sera positive with much higher titres than the others. The variability was not obviously related to the subtype used as antigen and for generation of the polyclonal rabbit and guinea-pig reagents. For example, most labs used O Manisa reagents for serotype O, but there were nevertheless, considerable quantitative and qualitative differences in results. Eight laboratories provided results from a serotype O specific SPCE test, after using the original or modified method of Mackay et al. (2001), which have cut-offs of 30% and 60% respectively (Table 5). One lab evaluated a new ELISA kit from Lelystad and another used a SPBE. Some labs also reported results for prototype SPCE based on type A specific or type Asia 1 specific reagents (Table 5). The strong positive sera were mostly found positive, but the weak positive sera sometimes gave results just below the cut off level. The cut-off sera were mostly scored negative. More sera were scored positive using the original Mackay method and cut-off of 30%, than using the modified method and the 60% cut-off. Some labs also tested for cross-serotype reactivity in VNT and structural protein ELISAs (data not shown). Results from Lab 1 for VNT and lab 2 for LPBE, indicated no or negligible cross-serotype reactivity by VNT and LPBE respectively. SPCE results from Pirbright and lab 2 showed weak cross-reactivity, with the A Iran 96 strong positive serum scoring positive or near-positive in the test using O Manisa reagents. The CEDI serotype O test evaluated by lab 4 scored all of the heterologous strong positive sera as positive, and also gave borderline reactions with some of the weak positive heterologous sera. In different NS tests, the sera obtained from vaccinated animals (serotypes A and Asia 1) were consistently negative. All NS tests scored the strong positive O SKR serum as positive. In-house NS tests gave a positive result with the weak positive O SKR serum, whilst it was scored positive, weak positive or negative by the Chekit-FMD-3ABC ELISA (Bommeli) in the three labs that evaluated this kit. Only the in-house blocking 3ABC ELISA of lab 8 scored the O SKR cut-off serum as positive. 227

3 Discussion It was not always clear how closely laboratories had followed the methods described in the OIE Manual of Diagnostic Tests. For example, a range of unusual serum dilutions was used to calculate end-point titres. A further point of ambiguity was how end-point titres had been calculated from serum dilutions, and whether initial or final serum dilutions had been used for this purpose. This needs to be standardised and it is recommended that final dilutions are used, as described in the OIE Diagnostic Manual. As expected, consistent results were obtained for the negative and the strong positive sera. However, the weak positive sera scored positive or inconclusive in VN tests with homologous viruses. The cut-off sera scored inconclusive or negative in the equivalent tests. Similar, though variable, results were obtained with ELISA, indicating that these sera are too weak for use as reference standards. There was an indication of reduced sensitivity if a heterologous strain of virus was used in the VNT, for example using O Manisa in place of O SKR, or A22 in place of A Iran 96. In the case of the A Iran 96 weak positive serum the mean arithmetic titre fell from 1:148 with the homologous virus to 1:13 with A22. The effect of test strain selection on the LPBE and SPCE was difficult to evaluate from these results, although in the case of LPBE, use of A22 instead of A Iran 96 did appear to impair sensitivity. The most inconsistent results were obtained with the A Iran 96 sera. In some labs, the titres obtained for VNT and LPBE were much higher than in others, such that even the cut-off serum was sometimes clearly positive. The O SKR sera should be suitable reference standards for NS serology once strengthened. The definitions for the weak and cut-off sera should be improved and explicitly related to the purpose for which testing is performed. It is suggested that the weak positive serum should represent a minimum standard for detection in any test used for herd-based serosurveillance. It should be primarily designed by reference to the virus neutralization test (VNT) using homologous virus. The cut-off for the VNT when screening on a herd basis is 1:45. Therefore the weak positive serum should have a mean VNT value of 1:64. The cut-off serum should represent a minimum standard for detection in any test used for individual animal certification. It should be primarily designed by reference to the virus neutralization test (VNT) using homologous virus. The cut-off for the VNT when screening on an individual animal basis is 1:16. Therefore the cut-off serum should have a mean VNT value of 1:32. Conclusion More explicit instructions should accompany ring test reagents, to improve standardisation of approaches and interpretations. It is difficult to produce weak positive and cut-off sera that behave similarly in different tests. Homologous VNT should be the gold standard, but preferably, a range of sera from different animals should be available in order to select standards with the broadest range of acceptable qualities. 228

4 Initial results with the SPCE and NS tests are promising and need to be further evaluated. It is proposed that the weak and cut-off positive sera need to be made stronger, so that the weak positives are consistently positive by most labs by VNT, and the cut-off sera are consistently inconclusive positive (i.e. not negative). Acknowledgements Thanks are due to all of the labs and their staff, who participated in this collaborative exercise. References Mackay DKJ, Naci Bulut A, Rendle T, Davidson F, Ferris NP. (2001). A solid-phase competition ELISA for measuring antibody to foot-and-mouth disease virus. Journal of Virological Methods 97,

5 Table 1. Dilutions of strong positive original sera to make reference sera Original sera Dilution of original sera to make: Strong +ve serum Weak +ve serum Cut-off serum O SKR 1:2 1:10 1:42 (34 days after infection) A Iran 96 1:2 1:24 1:90 (39 days after 1 st vaccination) Asia 1 Shamir 1:2 1:18 1:80 (47 days after 1 st vaccination) Table 2. Participants Dr Roland Silber, Vienna, Austria Dr Karl Sorensen, Lindholm, Denmark Dr Emiliana Brocchi, Brescia, Italy Dr Luis Romero, Madrid, Spain Dr Naci Bulut, Ankara, Turkey Dr Kris de Clercq, Ukkel, Belgium Dr Bernd Haas, Tuebingen, Germany Dr Aldo Dekker, Lelystad, The Netherlands Dr Barabara Thuer, Mittelhausern, Switzerland 230