Vaccine 26 (2008) 65776586

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Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Updating of the correlation between lpELISA titers and protection from virus challenge for the assessment of the potency of polyvalent aphtovirus vaccines in Argentina
Eduardo Maradei b,1 , Jos La Torre a,1 , Blanca Robiolo a , Jorge Esteves b , Cristina Seki a , Andrea Pedemonte b , Marcela Iglesias a , Ricardo DAloia b , Nora Mattion a,
a b

Centro de Virologa Animal, Instituto de Ciencia y Tecnologa Dr. Cesar Milstein, CONICET, Saladillo 2468, C1440FFX, Ciudad de Buenos Aires, Argentina Servicio Nacional de Sanidad y Calidad Agroalimentaria SENASA, Av. Fleming 1653, B1640CSI, Martnez, Argentina

a r t i c l e

i n f o

a b s t r a c t
Routine vaccination campaigns are carried out in Argentina twice a year, involving more than 100 million doses of foot-and-mouth disease (FMD) vaccine. Although the challenge test in cattle has not been totally replaced for the assessment of FMD vaccine potency, Argentine Animal Health authorities have used an indirect alternative method based on specic correlation studies of protection against podal generalization (PPG) tests performed in cattle with a validated liquid phase blocking ELISA (lpELISA). The change of vaccine formulations that took place after the 20002001 outbreaks, generated a gap in the correlation between lpELISA titers and PPG for the new FMD virus strains. A reappraisal of the correlation between lpELISA titers measured at 60 dpv and virus challenge by the PPG method at 90 dpv, performed for the four virus strains presently included in the Argentine vaccine is presented in this work. The data were obtained from 40 bovine challenge trials (647 sera) performed using exclusive batches of commercial vaccine from the year 2001 to January 2008 for A24/Cruzeiro, A/Argentina/2001, O1/Campos and C3/Indaial FMD virus strains. Curves of percentage of expected protection (EPP) versus lpELISA titers were obtained by logit regression for A/Argentina/2001, O1/Campos and C3/Indaial strains, but not for A24/Cruzeiro strain. The concordance between the direct and indirect tests using an EPP cut off value of 75% (82%, kappa = 0.62), in agreement with data originating from many years of vaccine control in Argentina, remarks the relevance of the acceptance of indirect alternatives to in vivo potency testing. 2008 Elsevier Ltd. All rights reserved.

Article history: Received 10 August 2008 Received in revised form 12 September 2008 Accepted 15 September 2008 Available online 1 October 2008 Keywords: Foot-and-mouth disease IpELISA Vaccine potency Alternative potency test

1. Introduction Foot-and-mouth disease (FMD) is one of the economically most important diseases that affect livestock [13]. Inactivated foot-andmouth disease vaccines are used in many parts of the world, and even in countries where vaccination is not currently used, concentrated inactivated FMD virus antigens are also kept in strategic reserves (antigen banks), which can be rapidly formulated into vaccines during an emergency [4]. The measurement of the potency of the vaccines is critical for the control and eradication of FMDV. Potency testing of inactivated vaccines is performed in the target species using direct or indi-

Corresponding author. Tel.: +54 11 4686 6225; fax: +54 11 4686 6225. E-mail addresses: nmattioncevan@centromilstein.org.ar, mattion@bertel.com.ar (N. Mattion). 1 The rst two authors contributed equally and kindly request a joint rst coauthorship. 0264-410X/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2008.09.033

rect methods. Up until now, the Gold Standard test is the in vivo challenge of primo-vaccinated animals. There are two direct methods commonly in use: 50% protective dose (PD50 ) and protection against podal generalization (PPG). The PD50 test consists in a measurement of potency by testing the vaccine in groups of at least ve cattle inoculated with different dose volumes of vaccine, so that potency can be expressed in terms of 50% protective doses [5]. The PPG method, in use in Latin America, is described in this work. It has been reported that observation of PD50 results indicated a lack of doseresponse relationship in a large number of tests, which complicated the interpretation of the results [68]. The PPG method, where the vaccine is used undiluted, proved to be highly reliable [9]. However, the challenge methods per se have practical and logistical problems, and many disadvantages from the perspective of animal welfare and biosafety. In most countries, ofcial animal health services as well as the Ofce International des Epizooties (OIE) experts have supported the use of alternative testing methods provided that a statistical correlation can be established between

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them and the challenge test in the target species. This is also the case in Argentina, where the in vivo PPG test is actually being implemented only for vaccine registration or for the introduction of new strains into an already registered vaccine (vaccine updating). It is generally accepted by FMD research workers that there is a good correlation between the virus neutralization (VN) antibody titers of primo-vaccinated cattle and their protection from virus challenge [1014], but several ELISAs have also been validated for this purpose and have been found to be at least as reliable and precise than the VN tests [13,1518]. In addition, ELISA can be used with inactivated antigens outside high security laboratories, the data can be obtained earlier and they are more friendly for validation and automatization. Moreover, ELISA results were found signicantly more reproducible [15,16]. In Argentina, we have reported several extensive studies using a liquid phase blocking ELISA (lpELISA) for the evaluation of protective immunity in cattle vaccinated with commercial oil-adjuvanted vaccines. In 1993, potency tests involving 1634 animals were reported [18], taking advantage of the assay of 102 batches of commercial vaccine, representing approximately 100 million doses, made over a period of 14 months in the years 1991 and 1992. Results showed that lpELISA serum titers directly correlated with the percentage of protected animals in vaccinated cattle challenged with FMDV strains O1/Caseros, A/Argentina/79, A/Argentina/87 and C3/Argentina/85, which represented the prototype strains used for vaccine production. In 1995, Robiolo et al. [19] extended the previous study presenting the results using lpELISA serum titers of 3920 vaccinated cattle, challenged by the PPG method with the same strains as in the previous study. In this case, two independent numerical coefcients were introduced. One of them was the lowest expected protection (LEP) calculated from the serum lpELISA titers at 60 days post vaccination (dpv) of animals challenged with virus at 90 dpv. The LEP evaluation was highly specic (i.e. it was able to predict vaccine failure in 100% of the cases), although its ability to predict the challenge approval (sensitivity) comprised only 65% of the vaccines that passed the PPG trial. The sensitivity was improved by introducing an alternative coefcient (Ro) capable of predicting the PPG approval of 90% of the vaccines, maintaining acceptable safety levels (87% specicity) [19]. Later on, these studies were extended by 435 vaccine series involving a total of 7390 vaccinated/challenged bovines, conrming the previously reported results (unpublished data). With the reintroduction of FMDV in Argentina in the years 20002001 several vaccine matching trials were performed for the emerging strains and there was a total change in vaccine composition [20]. The regional strains A24/Cruzeiro and O1/Campos were introduced together with the emerging local strains A/Argentina/2000 and A/Argentina/2001. A preliminary correlation between serological response and protection from challenge with O1/Campos and A/Argentina/2001 FMD virus strains was published with data derived from experimental and commercial vaccine challenge trials [21]. To establish a correlation of serology with challenge, data from 156 and 138 cattle vaccinated and challenged by intradermolingual inoculation with A/Argentina/2001 and O1/Campos FMDV strains, respectively, were analyzed in order to validate the application of indirect vaccine potency assays and assessment of vaccination efciency [21]. Recently, restricted in vivo trials were performed for vaccine matching purposes using a well-characterized A24/Cruzeiro monovalent experimental vaccine in six homologous (A24/Cruzeiro) and four heterologous (A/Argentina/2001) virus challenge trials performed at 30 days post vaccination [22]. In this work, a correlation of lpELISA titers of sera taken at 60 dpv from animals challenged by PPG at 90 dpv with each one of the

four strains present in the Argentine vaccine currently in use (A24 Cruzeiro, A/Argentina/2001, O1 Campos and C3 Indaial) is reported. The study was performed using exclusive batches of commercial vaccine and showed that the lpELISA in use in Argentina offered a high degree of reliability and safety in predicting the potency of vaccines in the absence of live virus challenge. 2. Materials and methods 2.1. Viruses and vaccines The four FMDV strains used in this study were A24/Cruzeiro/Brazil/55 (A24/Cruzeiro), O1/Campos/Brazil/58 (O1/Campos) and C3/Indaial/Brazil/71 (C3/Indaial) (origin: Pan American Centre for Foot-and-Mouth Disease (PANAFTOSA)), and A/Argentina/2001 (A/Arg/01), all of them provided by the Argentine Animal Health Service (SENASA). The serum samples were also provided by SENASA and originated from ofcial potency trials for polyvalent batches of commercial vaccine produced by different manufacturers. The formulation of the vaccines tested was not exactly the same, considering that the FMDV strain C3/Indaial was incorporated into the Argentine vaccines in the year 2005 in place of A/Argentina/2000 strain [20,21]. 2.2. Protection against podal generalization The vaccine potency trials considered in this study were performed by SENASA from the year 2001 up to January of the year 2008. Vaccines were from commercial origin, presented to SENASA either for registration or serial control. A total of 40 bovine trials were performed. In each trial the animals were challenged with only one virus strain at a time, as summarized in Table 1. PPG tests were conducted according to SENASA Resolution no. 351/06 [23], as previously described [20-22]. Briey, 16 individually ear-tagged cattle, negative for FMDV antibodies, were vaccinated intramuscularly in the upper part of the neck with a full cattle vaccine dose (2 ml). Two unvaccinated control animals were included in each potency trial. Ninety days post vaccination, 16 vaccinated animals and both unvaccinated controls were challenged by inoculating intradermally 104 suckling mouse lethal doses 50% (SMLD50 ), equivalent to 104 BID50 (unpublished data), of challenge virus into four different sites on the upper surface of the tongue (0.25 ml per site). Seven days post challenge (dpc) all animals were clinically checked for FMDV-induced lesions on the feet. PPG was calculated as the number of vaccinated protected animals (i.e. absence of FMDV-induced lesions at the feet) divided by the total number of vaccinated animals 100. The trial was considered valid if both non-vaccinated control animals showed FMDV-induced lesions on at least one foot. According to Resolution no. 351/06 [23], a vaccine batch is approved for
Table 1 PPG trials carried out for commercial vaccines in Argentina. Challenge strain A24/Cruzeiro A/Arg/01 O1/Campos C3/Indaial Total
a b c d

No. of trials 6 11 16 7 40

No. of serum samples 94a 177b 258c 118d 647

Two animals were discarded for outliers. 17 animals were challenged in one of the trials. 17 animals were challenged in two of the trials. 17 animals were challenged in six of the trials.

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Fig. 1. Design of the lpELISA microplates. B: Blank; 100%: antigen controls (100% absorbance); 116: serum samples; C1C6: serum controls. C: negative control.

licensing if at least 12 out of the 16 vaccinated animals are found to be protected (i.e. 75% PPG). A vaccine batch must be retested if 1011 vaccinated cattle are protected against challenge (i.e.: 62.568.8% PPG), and a vaccine batch is rejected if only 9 or less vaccinates show absence of lesions on the feet (i.e.: lower than 62.5% PPG). The cattle used originated from Patagonia (Argentina), a region ofcially recognized by the OIE as free from FMD without vaccination [24]. The animals selected were steers, between 24 and 30 months of age and weighing 280350 kg, healthy, with a good nutritional status and free of parasites. Prior to the study, all cattle were bled and the absence of anti-FMDV antibodies was checked using lpELISA [18,19], and 3ABC-ELISA [25] to conrm the absence of prior contact to structural and non-structural FMDV proteins. Animals were then bled at 30, 60 and 90 dpv. The serology used for correlation with PPG corresponded to the 60 dpv sera. The challenge with live virus was carried out in the BSL3A facilities of the Instituto Nacional de Tecnologa Agropecuaria (INTA) located in Castelar, province of Buenos Aires, according to biosecurity and animal welfare federal regulations [23]. 2.3. Liquid phase blocking ELISA The assay was originally adapted from Hamblin et al. [2628] and was carried out with modications [1821]. Serum samples were tested in 96-well plates in fourfold dilutions (Fig. 1). Four sample dilutions (from 1:32 to 1:2048) were incubated overnight at 4 C with a pre-titrated dose of the corresponding virus strain in a saline buffer liquid phase. The mixtures were then incubated for 1 h at 37 C on wells coated with FMDV strain specic rabbit polyclonal antibody, where the virus that did not react with the bovine serum in the previous step was trapped. After a washing step (ve times with PBS containing 0.05% Tween, v/v) a second incubation was performed with a saturating concentration of a pool of monoclonal antibodies (MAbs) specic for each of the virus strains being tested. Goat anti-mouse IgG conjugated with horseradish peroxidase (Jackson InmunoResearch, USA) was added, and colour development was obtained after addition of the substrate/chromophore mixture (H2 O2 /ABTS (2,2-azino-bis3-ethyl-benzothiazoline-6-sulfonic acid diammonium salt, Sigma,

USA). The optical densities (OD) readings were measured using an automatic microplate reader (Bio-Rad, Hercules, CA) at a wavelength of 415 nm. Six control sera of known titers (reference sera of high, medium and low titers) were assayed simultaneously as internal standards in each ELISA plate in three twofold dilutions from 1:32 to 1:128. Two negative control sera (unvaccinated animals) were tested in one dilution (1:16). Eight wells were used for antigen concentration control (100% reactivity) and two wells were used as reaction blanks without virus antigen and without serum. Antibody titers were expressed as the reciprocal log10 of serum dilutions giving 50% of the absorbance recorded in the antigen control wells (OD50% ). Calculations and validation of each plate were performed using proprietary software (Robiolo et al., unpublished). For validation of each lpELISA plate, the following performance criteria were applied. 2.3.1. Blank The average absorbance of the two blanks should be <0.300. This value was established considering that the mean blank value plus 3 S.D. is always lower than 0.300 for the four FMDV strains. The blank OD was subtracted from the OD value of every well. 2.3.2. Antigen control The OD of each replicate should be >0.750 and <1.950, not differing from each other in more than 0.300 OD. At least seven out of eight replicates had to comply with this condition. The average of the eight wells (or seven valid wells) was calculated and OD 50% was established (OD50% ). The antigens used as standards were stored in 50% glycerol and 0.1% sodium azide. 2.3.3. Serum controls The titer of each control serum was established as described above. The dilutions used corresponded to the linear part of the curves obtained representing OD readings versus the log of the dilutions used in the assay. The coefcients of correlation (r) were calculated. r values should be 0.90 for each control serum. Titers of control sera should not differ in more than 0.200 from the reference values. A correction factor (f) was applied if at least four out of six control sera presented differences of the same sign, to account for eventual errors in virus concentration on the plates, which is

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a critical assay parameter. f values applied were as follows: If the mean of the differences from the reference values was lower than 0.059 there was no correction; between 0.060 and 0.109, f = 0.05; for 0.1100.159, f = 0.10; for 0.1600.205, f = 0.15. After correction, when corresponded, serum titers should not differ in more than 0.13 from the reference values. Four out of six control sera should comply with all the conditions cited above in order to validate each plate. 2.3.4. Serum samples Titers were calculated for each sample and the correction factor f was applied when corresponded. The curves representing OD values versus log10 of serum dilutions of the 16 serum samples were generated using the software. The graphic had to show sigmoid shaped curves typical of doseresponse in a parallel array in order to validate each plate. This verication was done visually. 2.4. Validation of the lpELISA Intra-plate and intra-day repeatability were estimated previously (data not shown). The intra-laboratory repeatability precision was estimated with data obtained by different analysts, selecting at random one lpELISA plate from 15 different days, for each of the six control serum of every virus strain being tested (24 control sera), over a 3 months period (Lab #1). Other estimations of intermediate precision were performed in two other laboratories. In Lab #2, belonging to SENASA the same procedure as before was performed for 14 different days over the period of one year. In the Lab #3, belonging to our Institution but in a different location, 3443 independent testing of the six control sera for each vaccine strain were carried out over a period of 5 months. Standard deviations (S.D.) and coefcients of variation (CV%) were calculated [29]. Reproducibility was estimated with 14 determinations of lpELISA titers in independent assays of the 24 control sera in each of the three mentioned laboratories. S.D.s and CVs% between the three labs were calculated. The specicity data of the lpELISA for each vaccine strain was established, based on assays of negative sera obtained from bovines of the Patagonia region. In this case only one dilution (1:16) of the serum samples was assayed. Animals were considered as negatives if antibody titer values were 1.3. Thirty-two control charts [29] were used to monitor the performance of the assay in time (i.e. one per each six control sera of every serotype, four for each 100% antigen OD and four for each blank). Studies performed to establish the regression model between the titers obtained in CEVAN lpELISA and the PANAFTOSA ELISA were performed as follows. Forty sera of each FMDV strains A24/Cruzeiro, C3/Indaial and O1/Campos, corresponding to 10 different antibody levels were titrated by both assays. Each serum was repeated 16 times, and the mean, maximum and minimum titers were determined. Using previous results from the Rio de La Plata Basin Subproject (CEE/Cuenca del Plata/CPFA/OPS [30]) regarding the correlation of PANAFTOSA ELISA titers with PPG, the percentage of expected protection (EPP) for every CEVAN lpELISA titer in the PANAFTOSA logit curves could be established. There were no tests available for A/Arg/01 strain in PANAFTOSA. Further collaborative studies were performed analyzing 621 serum samples with both tests in several public and private laboratories of Argentina and South America. 2.5. Statistical analysis and correlation with PPG The EPP for each FMDV strain was calculated by comparing the levels of lpELISA antibody titers from sera collected at 60 dpv with results from cattle protection in challenge tests (PPG) at 90 dpv.

The statistical model used to nd the correlation between the binary variable (protected/unprotected) and the continuous variable (lpELISA titer) was the logistic regression (Logit), using the formula: Logit(p) = ln(p)/[1 ln (p)] = + xi , where x is the lpELISA titer and p is the probability of the event protected for a given x titer. The probabilities were calculated using the logit estimators ( and ) derived from the database obtained from the trials. The values of p, which indicates the probability of the model adjustment (tting), were calculated. For the calculation of the theoretical EPP the formula used was: EPP% = Exp(Logit)/(1 + Exp(Logit) [31]. 2.6. Concordance between vaccine status by PPG or EPP The EPP for each individual vaccine was calculated with the logit regression curves using the mean of the lpELISA titers of the 16 animals involved in each PPG trial. These values were used for the establishment of concordance between both in vivo and in vitro methodologies for vaccine approval. 3. Results 3.1. PPG trials The performance of 40 commercial vaccines in the PPG trials was as follows: 30 approved, 7 were rejected and 3 were sent for retesting. All 647 sera from vaccinated animals and sera from the unvaccinated controls collected at 0, 30, 60 and 90 dpv, were assayed by lpELISA for each FMDV strain present in the vaccines. However, only the 60 dpv titers were considered to study the correlation with PPG. The monitoring of the evolution of the lpELISA titers upon time constitutes an overall control of the trials and of the animals serological status. Usually, most animals seroconverted by 14 dpv. Then, serum titers continue to increase very slowly and most of them reach a plateau or even start to decrease by 60 dpv. In the case of high potency vaccines serum titers continue to increase even after 90 dpv (data not shown). All unvaccinated control animals remained negative during the trial and seroconverted at 7 dpc (data not shown) showing clinical signs of the disease (100% assay sensitivity). Although only 16 animals per trial are considered for the calculation of PPG and EPP, it is usual to vaccinate17 animals in order to prevent eventual losses. Then, the animal with the lowest titer is discarded. Given the low number of trials that have been performed, animal number 17 was also challenged in many trails and although it was not considered for potency assessment, the lpELISA titer and protection status were included in the database (Table 1). 3.2. lpELISA validation The lpELISA described in this report is ofcially used in Argentina as indirect potency test for FMD vaccines and was thoroughly validated. Data on intermediate precision of the test was obtained from different laboratories (Table 2). CV% values were in all cases <10% when performed by different analysts in different days, which was the acceptance criteria [29]. lpELISA reproducibility was assessed from 14 determinations of the 24 control sera carried out in the three mentioned labs (Table 3) and CV% values were found to be <10%. The diagnostic specicity of the lpELISA for each vaccine strain was established, based on assays of negative sera obtained from bovines of the Patagonia region (Table 4). For the selected cut off corresponding to a titer 1.3, the specicity of the assay for each FMD virus strain was established as follows: 99.8% for O1/Campos

E. Maradei et al. / Vaccine 26 (2008) 65776586 Table 2 Intermediate precision of lpELISA assessed in three different laboratories for 24 control sera. Strain Lab #1 Mean A24/Cruz. C #1 C #2 C #3 C #4 C #5 C #6 A/Arg/01 C #1 C #2 C #3 C #4 C #5 C #6 O1/Cam C #1 C #2 C #3 C #4 C #5 C #6 C3/Ind C #1 C #2 C #3 C #4 C #5 C #6 2.06 1.85 1.68 2.14 1.90 1.68 1.97 1.76 1.55 2.02 1.85 1.72 2.26 1.96 1.83 2.30 2.00 1.82 2.12 1.83 1.73 2.04 1.83 1.60 S.D. n = 15 0.07 0.07 0.10 0.09 0.04 0.01 n = 15 0.08 0.09 0.12 0.06 0.10 0.08 n = 15 0.06 0.05 0.05 0.12 0.03 0.04 n = 15 0.07 0.04 0.07 0.07 0.07 0.05 CV (%) 3.59 3.54 6.20 4.03 2.37 5.99 3.97 5.14 7.65 3.05 5.24 4.66 2.82 2.41 2.92 5.09 1.54 2.04 3.32 2.37 3.82 3.39 3.65 3.31 Lab #2 Mean 2.11 1.90 1.72 2.15 1.92 1.78 1.95 1.79 1.53 1.99 1.79 1.57 2.23 1.99 1.85 2.28 2.02 1.87 2.17 1.89 1.77 2.06 1.83 1.60 S.D. n = 14 0.12 0.12 0.10 0.11 0.08 0.09 n = 14 0.08 0.10 0.14 0.09 0.07 0.12 n = 14 0.15 0.08 0.09 0.13 0.09 0.07 n = 14 0.12 0.09 0.08 0.09 0.06 0.10 CV (%) 5.50 6.21 6.05 5.12 4.44 5.17 4.13 5.61 9.50 4.37 3.94 7.42 6.71 3.90 4.83 5.82 4.58 3.49 5.41 4.62 4.33 4.37 3.45 6.50 Lab #3 Mean 2,06 1.85 1.66 2.14 1.93 1.72 1.98 1.78 1.55 2.07 1.86 1.63 2.20 1.94 1.80 2.28 1.98 1.81 2.22 1.86 1.73 2.07 1.81 1.59 S.D. n = 43 0.08 0.04 0.07 0.11 0.14 0.09 n = 37 0.06 0.05 0.08 0.08 0.05 0.07 n = 43 0.10 0.06 0.05 0.13 0.06 0.06 n = 34 0.12 0.06 0.07 0.10 0.06 0.09

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CV (%) 3.88 2.38 4.35 5.12 7.10 5.41 2.83 2.95 5.01 3.84 2.78 4.18 4.52 3.11 2.99 5.77 3.25 3.20 5.61 3.08 4.12 4.66 3.48 5.73

n: number of assays; S.D.: Standard deviation; CV: Coefcient of variation.

(n = 1045); 99.9% for A24/Cruzeiro (n = 799); 99.7% for A/Arg/01 (n = 779) and 99.6% for C3/Indaial (n = 785). Thirty-two control charts were used for monitoring the performance of the assay in time. The values obtained for the serum controls usually remained within 2 S.D. Three of these charts, representing one control serum of each serotype, are shown in Fig. 2. The titers obtained are plotted as points, the dotted line represents the mean titer and the solid lines represent 1 S.D., 2 S.D. and 3 S.D. from the mean. The mean and S.D. were initially found after 15 determinations of each serum (C #5 for A/Arg/01, C #2 for O1/Campos and C #3 for C3/Indaial (mean S.D.: 1.85 0.097, 1.96 0.047 and 1.73 0.066, respectively)). In this case, the chart represents 50 independent runs of the assay, performed within 5

Table 4 Diagnostic specicity of lpELISA for FMDV strains A24/Cruzeiro, A/Arg/01, O1/Campos and C3/Indaial. Strain O1/Campos (n = 1045) lpELISA titers 0.9 1.0 1.1 1.2 1.3 1.4 1.5 0.9 1.0 1.1 1.2 1.3 1.4 1.5 0.9 1.0 1.1 1.2 1.3 1.4 1.5 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.5 Freqa 987 34 16 4 2 2 0 695 64 24 12 3 1 0 627 62 52 27 9 2 0 621 121 26 11 3 1 2 0 % 94.4 3.3 1.5 0.4 0.2 0.2 0.0 87.0 8.0 3.0 1.5 0.4 0.1 0.0 80.5 8.0 6.7 3.5 1.2 0.3 0.0 79.1 15.4 3.3 1.4 0.4 0.1 0.3 0.0 Acc %b 94.4 97.7 99.2 99.6 99.8 100 100 87.0 95.0 98.0 99.5 99.9 100 100 80.5 86.4 95.1 98.6 99.7 100 100 79.1 94.5 97.8 99.2 99.6 99.7 100 100

A24/Cruz. (n = 799)

Table 3 Reproducibility of lpELISA assessed in three different laboratories. Strain A24/Cruz. C #1 C #2 C #3 C #4 C #5 C #6 A/Arg/01 C #1 C #2 C #3 C #4 C #5 C #6 Meana 2.07 1.87 1.70 2.15 1.93 1.74 1.98 1.79 1.57 2.05 1.83 1.60 S.D. 0.09 0.08 0.09 0.11 0.14 0.08 0.07 0.07 0.11 0.13 0.08 0.10 CV (%) 4.37 4.34 5.20 4.96 7.14 4.49 3.55 4.13 6.84 6.32 4.10 6.28 Strain O1/Cam C #1 C #2 C #3 C #4 C #5 C #6 C3/Ind C #1 C #2 C #3 C #4 C #5 C #6 Mean 2.22 1.96 1.82 2.32 2.01 1.83 2.19 1.87 1.74 2.06 1.81 1.58 S.D. 0.11 0.06 0.06 0.16 0.08 0.05 0.10 0.07 0.06 0.08 0.05 0.09 CV (%) 4.89 3.17 3.32 6.70 3.77 2.97 4.77 4.00 3.41 4.07 2.93 5.56 A/Arg/01 (n = 799)

C3/Indaial (n = 785)

S.D.: standard deviation; CV: coefcient of variation. a Mean of 42 determinations (14 in each Lab).

n: number of serum samples tested. a Frequency. b Accumulated frequency.

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Fig. 2. Control charts representing one control serum of each FMDV serotype (C #5 for A/Arg/01, C #2 for O1/Campos and C #3 for C3/Indaial (mean S.D.: 1.85 0.097, 1.96 0.047 and 1.73 0.066, respectively)). The charts represent 50 independent runs of the assay, performed within 5 months. ( ) lpELISA titer after application of the correction factor. The dotted lines represent the mean titer (found after 15 determinations of each serum) and the solid lines represent 1 S.D., 2 S.D. and 3 S.D.

months. The CV% were 5.24%, 2.41% and 3.82%, respectively, for the serum controls mentioned above. Parallel determinations of serum titers using the CEVAN lpELISA and the PANAFTOSA ELISA, performed on the same set of sera at

PANAFTOSA (Brazil) showed a good correlation for the regional FMDV strains A24/Cruzeiro, O1/Campos and C3/Indaial, with R2 values of 0.9804 for O1/Campos, 0.7851 for A24/Cruzeiro, and 0.9903 for C3/Indaial. The range of variation was satisfactory, presenting

Fig. 3. Correlation of titers obtained using the CEVAN lpELISA and PANAFTOSA ELISA, for O1/Campos, A24/Cruzeiro and C3/Indaial FMDV strains. (A) The data shown was collected from assays performed in CEVAN, SENASA and PANAFTOSA laboratories. (B) The data shown was collected from assays performed in parallel with both ELISA kits by technicians belonging to different public and private laboratories following the protocols of CEVAN and PANAFTOSA.

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good parallelism and the expected tendency for the different titer levels (not shown). Statistically, the capacity of both assays to differentiate sera with different antibody levels was demonstrated. Dispersion diagrams are shown in the upper panel of Fig. 3 for each FMD virus type (O1/Campos, C3/Indaial and A24/Cruzeiro). A linear relationship between titers determined with both assays can be observed. Further collaborative studies were performed analyzing 621 serum samples with both tests. Data was collected from several public and private laboratories of Argentina and South America (Brazil and Colombia). The results are shown all together in the lower panels of Fig. 3, with the corresponding R2 values for each strain, displaying a good correlation of the titers obtained with both assays at different laboratories. Preliminary data from assay robustness was obtained running the assay with control antigens heated at 56 C for 2 min, in order to degrade 140S virus particles into 12S protomers. Untreated antigens were used as controls. All the serum controls were validated similarly in both cases (data not shown). A similar experiment was performed after storage of the antigens at 37 C for 40 days. We are carrying out systematic experiments in order to further study the stability of the antigens and its impact on the performance of the assay. 3.3. Correlation between protection against challenge and lpELISA titers in vaccinated cattle Data on in vivo protection (PPG) and lpELISA titers for each virus strain analyzed (A/Arg/01, O1/Campos, C3/Indaial A24/Cruzeiro) are shown in the four left panels of Fig. 4(AD). In each case the number of sera analyzed is shown. On the left side of the gures, the PPG data is depicted as an histogram ordered per xed lpELISA titer interval. The bar sizes account for the percentages of protection corresponding to a specic titer interval. The total number of animals whose serum titers are included in each titer interval is shown at the top of each bar. A close correlation can be observed between the serum lpELISA titers and the percentage of in vivo protected animals for A/Arg/01 and O1/Campos FMDV strains, whereas a good but less close correlation was established for C3/Indaial strain, for which only seven PPG trials were carried out. Using these databases, the logit estimators and were calculated and were found highly signicant (p < 0.01) for the four FMDV strains (Table 5). The tting of the models, given by the value of pvalue (probability of model adjustment) was obtained for three of the FMDV strains as follows: A/Arg/01 = 0.8186, O1 Campos = 0.8819 and C3 Indaial = 0.9768. In the case of A24/Cruzeiro strain, the regression could not be obtained with the data available in this work (p > 0.05). The theoretical EPPs were calculated from the logit transformation with the formula EPP% = Exp(Logit)/(1 + Exp(Logit). In the right side of each panel of Fig. 4, the EPP curves for lpELISA titers extended from 0.60 to 4.1 obtained by logit regression are shown for A/Arg/01, O1/Campos and C3/Indaial FMDV strains. The cut off of 75% EPP is shown as a dotted line. Three tables were
Table 5 Parameters of logit transformation and model tting. Strain A/Arg/01 O1/Campos C3/Indaial A24/Cruz. 5.9346 4.9111 5.1759 1.7292 3.2019 2.8474 2.8965 1.8064 p-reg < 0.01 < 0.01 < 0.01 0.3728a p-tit < 0.01 < 0.01 < 0.01 0.0444 p-value 0.8186 0.8819 0.9768 0.9999

Table 6 Concordance of lpELISA and PPG for vaccine approval. PPG Approved lpELISA Approved Rejected Total Concordance Kappa
a b

Rejecteda 1b 9 10

Total 20 14 34

19 5 24 82% 0.62

Vaccines to be retested were considered as rejected. Status: retesting by PPG, approved by ELISA (EPP = 76%).

derived from this transformation were the EEP% value for every possible lpELISA titer for each of the strains can be found. These tables are not shown in this report for space problems but are available on-line as a Supplemental le. 3.4. Concordance of lpELISA titers and PPG performance for approval or rejection of commercial vaccines Vaccine status according to lpELISA titers was derived from the logit regressions curves shown in Fig. 4 for A/Arg/01, O1/Campos and C3/Indaial. Out of 34 PPG vaccine trials carried out for O1Campos, C3 Indaial and A/Arg/01, 20 vaccines were approved, 4 were rejected and 10 scheduled for retesting according to EPP values. In the case of A24/Cruzeiro strain (6 PPG trials), EPPs were derived from PANAFTOSAs correlation curve [30] and therefore they were not considered in this analysis. A two-way table was assembled with the data of both the in vivo trials and the in vitro assays (Table 6). For this exercise, vaccines sent for retesting were considered as rejected. Using a 75% cut off value for EPP, the concordance between both methods (PPG and lpELISA) was 82%, associated with a Kappa index of 0.62 (good concordance). Kappa values vary between 0 (not concordance) and 1 (full concordance) representing degrees of concordance excluding random events [32,33]. Nineteen out of 34 vaccines were approved by both methods while 9 out of 34 were rejected by both methods, 5 were approved by PPG but rejected by ELISA and 1 was approved by lpELISA and rejected by PPG (retesting). Fig. 5 shows the EPPs calculated from lpELISA titers of 34 batches of commercial vaccine series, where the status of approval or rejection by PPG challenge is represented by symbols. Vaccine series that did not pass PPG but may be admitted for a new potency test are represented as triangles. For an EPP cut off of 75% (dotted line) only one vaccine sent to retesting by PPG was approved based on their lpELISA titers (EPP = 76%). Although the EPP data was not derived from Argentine correlation curves, the potency data on vaccines formulated with A24/Cruzeiro strain are in agreement with the results mentioned above (gray lled circles in Fig. 5). 4. Discussion According to OIE regulations, indirect tests may be used to assess the potency of a vaccine provided that a statistical evaluation has established a satisfactory correlation between the results obtained by the indirect test on the relevant vaccine serotype and the potency test in cattle [5]. Although the most explored area in indirect potency methods has been the correlation between protection and neutralizing antibody, our group and others have had a long term experience in studying the correlation of potency based on ELISA titers either with experimental or commercial vaccines. As described in Section 1, one of the main strengths of this work is the fact that it was performed using commercial vaccine series

and : logit coefcients; p-tit: probability of signicant differences between titers of protected/not protected animals (should be <0.05); p-reg: probability of regression (should be <0.05). p-value: adjustment. a p-regression > 0.05.

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Fig. 4. Correlation of protection by PPG versus lpELISA titers. To the left, the histograms with data of in vivo percentage of protection (PPG) for each lpELISA titer interval are shown. The logit transformation curves are shown to the right for A/Arg/01 (A), O1/Campos (B), and C3/Indaial (C). No logit regression was obtained for A24/Cruzeiro strain (p > 0.05) (D) with the data generated in this work. EPP, expected percentage of protection; n, number of animal sera tested.

for which both data, lpELISA titers and PPG outcome, were available for particular FMDV strains. We had reported in two previous publications the correlation of direct and indirect potency tests (lpELISA) involving 5554 vaccinated/challenged bovines with the

Argentine strains (A/Arg/87, A/Arg/79, O1/Caseros and C3/Arg/85) [18,19]. Later on, these studies were extended to another set of 7390 vaccinated/challenged bovines with the same strains (unpublished data), reaching a total 12,944 vaccinated animals analyzed.

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Fig. 5. Concordance of lpELISA and PPG for 40 Argentine commercial vaccines, shown as vaccine status in PPG trials versus percent expected protection (EPP) calculated by lpELISA titers. ( ) Approved by PPG; () rejected by PPG; ( ) retesting by PPG. Gray area: EPP retesting area. EPP = 75% is depicted by a dotted line. The data corresponding to six A24/Cruzeiro vaccines approved by PPG whose EPP was calculated using PANAFTOSA logit regression curves [30] are shown by gray circles ( ).

Statistical analysis of this large database had been performed in the past in order to establish the correlations (EPP logit regression curves) used by SENASA for vaccine approval (Zanelli et al., unpublished). This work denitely denoted that the lpELISA was an indirect test which corresponded to protection in the target species in vivo, and was used in Argentina until FMD eradication in 1999. Vaccines manufactured after the year 2001 were composed of different virus strains and have been approved mostly by PPG carried out in BSL3A animal facilities, or by lpELISA titers based on the existing PANAFTOSA correlation curves for A24/Cruzeiro and O1/Campos strains and on preliminary data for A/Arg/01 strain [21]. This work reports a reappraisal of the correlation between in vivo challenge and indirect potency testing in the target species for FMDV strains considered (O1/Campos, C3/Indaial, A/Arg/01, A24/Cruzeiro). This is of great importance to countries with vaccination programs like Argentina. Multivalent commercial vaccine testing is performed nowadays by PPG challenge only for registration of new products (at least for the three rst commercial series) or in the case of an introduction of a new strain in an already existing vaccine. Another important consideration is that in multivalent FMD vaccines containing four different strains of FMDV it is not possible to undertake a challenge test with more than a single strain of virus at a time. Usually, sanitary authorities select one of them for a challenge trial and the other strains are tested by serology with lpELISA. The same criterion is applied for regular commercial vaccine series after the registration process is completed, where only sporadic at random in vivo tests are performed afterwards. The CV% of the lpELISA described in this work for intermediate precision and reproducibility (Tables 2 and 3) was below 10%, which is a suitable accepted value [29] and it showed diagnostic specicities over 99.5% and sensitivities of 100%, for the four studied FMDV strains (Table 4). Several further collaborative studies have been performed involving public and private laboratories of Argentina, Brazil and Colombia. Professionals of these labs have been capacitated at CEVAN in every case, before transference of the technique and its reagents (kits). The same assay was adapted for use in the screening of herd immunity status in the eld, using a single serum dilution (not shown in this work). In this work a statistically suitable correlation was established by logit regression for strains A/Arg/01, O1/Campos and C3/Indaial, but not for A24/Cruzeiro (regression p > 0.05). A24/Cruzeiro was the

strain less tested by PPG in commercial vaccine trials due to the fact that the emerging strain A/Arg/01 was prioritized in potency tests in vivo. It is worth mentioning that many experimental PPG tests have been carried out with A24/Cruzeiro, including the six recently described by Goris et al. [22]. C3/Indaial strain was also tested only in seven PPG tests because it was not incorporated into the vaccine until the year 2005. The strength of the correlations was shown in this work (Table 5 and Fig. 4). The logit estimators and were found highly signicant (p < 0.01) for three out of four FMDV strains and the tting of the model was given by the probability of signicant differences between titers of protected/not protected animals and the probability of regression, which were <0.05 in both cases for all strains except for A24/Cruzeiro. More tests in vivo involving commercial vaccines should be performed with A24/Cruzeiro challenge in order to establish a suitable correlation for this strain. When the analysis with a two-way table was applied (Table 6), it was clear that for the EPP cut off considered (75%) there were no vaccines rejected by PPG that were approved based on their lpELISA titers, except one vaccine that was approved by EPP with a PPG status of retesting. Two vaccines rejected and ve vaccines approved by PPG were considered for retesting by ELISA. The other two vaccines whose potency might be retested according to PPG (Fig. 5) were considered in the same status according to lpELISA titers. It is worth mentioning from the point of view of the validity of indirect methods, that although the serum titers for A24/Cruzeiro were determined with our lpELISA and the corresponding PANAFTOSA EPP correlation curve, the data obtained t into the same rationale: The six A24/Cruzeiro vaccines were approved by PPG and ve of them were also approved by ELISA, while the sixth was in the retesting area by lpELISA (Fig. 5). There is still a concern from the point of view of vaccine manufacturing companies based on the fact that ve vaccines that have passed PPG are considered for retesting by lpELISA. In this work we have used 75% EPP as an example, because it is the value being nowadays considered by SENASA in Argentina. For instance, lowering the EPP cut off to 70%, would mean that four more good vaccines are approved by lpELISA, but also two rejected vaccine and two that had been assigned for retesting by PPG would be approved by EPP. The cut off level to be used may be modied in order to meet the desired vaccine potency needed for control of FMD. In any case, sanitary authorities should establish the cut off values of the indirect test as it exceeds the scope of this work. Finally, we would like to emphasize that the reliability of lpELISA to evaluate the potency of FMD vaccines as well as its ease of adaptability for many different virus serotypes could lead in the near future to the complete replacement of the challenge procedures not only in Argentina but in other regions of the world, allowing the elimination of the massive use of in vivo challenge tests for regular vaccine testing and avoiding the use of live virus for that purpose. Efforts for the replacement of the in vivo potency tests performed for aphtovirus vaccines have been in course for a long time at several public and private laboratories [17,34] because of ethical and economical reasons. In the lpELISA described in this work, pools of well-characterized MAbs are used as detectors. This reagents are available for multicenter collaborative studies if they are required, where the best pool composition and working conditions can be set up for use with other FMDV strains. More than 80 MAbs have been developed in our laboratory (Seki et al., unpublished data) and a selection of the pools has been made for use with every different strain present in South America in the last 20 years. Many specic reagents (polyclonal and monoclonal) have been also reported by other labs [35,36]. Although international acceptance and production of reference materials might be a responsibility of the OIE, it will probably also depend on particular

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E. Maradei et al. / Vaccine 26 (2008) 65776586 [16] Amadori M, Archetti IL, Tollis M, Buonavoglia C, Panina GF. Potency assessment of foot and mouth disease vaccines in cattle by means of antibody assays. Biologicals 1991;19:1916. [17] Goris N, Willems T, Diev V, Merkelbach-Peters P, Vanbinst T, Van der Stede Y, et al. Indirect foot-and-mouth disease potency testing based on a serological alternative. Vaccine 2008;26:38709. [18] Periolo OH, Seki C, Grigera PR, Robiolo B, Fernandez G, Maradei E, et al. Large-scale use of liquid-phase blocking sandwich ELISA for the evaluation of protective immunity against aphthovirus in cattle vaccinated with oiladjuvanted vaccines in Argentina. Vaccine 1993;11:75460. [19] Robiolo B, Grigera PR, Periolo OH, Seki C, Bianchi T, Maradei E, et al. Assessment of foot and mouth disease vaccine potency by liquid-phase blocking ELISA: a proposal for an alternative to the challenge procedure in Argentina. Vaccine 1995;13:134652. [20] Mattion N, Knig G, Seki C, Smitsaart E, Maradei E, Robiolo B, et al. Reintroduction of foot-and-mouth disease in Argentina: characterization of the isolates and development of tools for the control and eradication of the disease. Vaccine 2004;22:414962. [21] Robiolo B, Seki C, Fondevilla N, Grigera P, Scodeller E, Periolo O, et al. Analysis of the immune response to FMDV structural and non-structural proteins in cattle in Argentina by the combined use of liquid phase and 3ABC-ELISA. Vaccine 2006;24:9971008. [22] Goris N, Maradei E, DAloia R, Fondevila N, Mattion N, Perez A, et al. Footand-mouth disease vaccine potency testing in cattle using homologous and heterologous challenge strains: precision of the Protection against Podal Generalisation test. Vaccine 2008;26:34327. [23] Animal Health Service (SENASA). Act no. 351/2006. In: Boletn Ocial no. 30.940, Argentina, July 5th, 2006 [available at http://infoleg.mecon.gov.ar/ infolegInternet/anexos/115000-119999/117636/norma.htm]. [24] Ofce International des Epizooties. List of Foot and Mouth Disease free countries. Resolution no. XXI [available at http://www.oie.int/eng/info/en fmd. htm?e1d6#Liste; updated August 9, 2007]. [25] Bergmann IE, Malirat V, Neitzert E, Beck E, Panizzutti N, Sanchez C, et al. Improvement of a serodiagnostic strategy for foot-and-mouth disease virus surveillance in cattle under systematic vaccination: a combined system of an indirect ELISA-3ABC with an enzyme-linked immunoelectrotransfer blot assay. Arch Virol 2000;145(3):47389. [26] Hamblin C, Barnett ITR, Crowther JR. A new enzyme linked immunosorbent assay (ELISA) for the detection of antibodies against FMDV. I. Development of a method for ELISA. J Immunol Methods 1986;93:11521. [27] Hamblin C, Barnett IT, Crowther JR. A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. II. Application. J Immunol Methods 1986;93(1):1239. [28] Hamblin C, Kitching RP, Donaldson I, Crowther JR. Barnett ITR Enzymelinked immunosorbent assay for the detection of antibodies against FMDV. III. Evaluation of antibodies after infection and vaccination. Epidemiol Infect 1987;99:73344. [29] Jacobson RH. Validation of serological assays for diagnosis of infectious diseases. Rev Sci Tech Off Int Epiz 1998;17:46986. [30] Pan-American Foot-and-Mouth Disease Center. Report on the Subproyecto para la correlacin de las tcnicas de control de potencia de las vacunas contra la Fiebre Aftosa en los pases de la Cuenca del Ro de la Plata. Rio de Janeiro, Brazil: PANAFTOSA; 1994. [31] Trautman R, Harris WF. Modeling and computer simulation approach to the mechanisms of foot-and-mouth disease virus neutralization assays. Scand J Immunol 1977;6:83141. [32] Cohen J. A coefcient of agreement for nominal scales. Educ Psychol Meas 1960;20:3746. [33] Fleiss JL, Cohen J, Everitt BS. Large sample standard errors of kappa and weighted kappa. Psychol Bull 1969;72:3237. [34] Barnett PV, Statham RJ, Vosloo W, Haydon DT. Foot-and-mouth disease vaccine potency testing: determination and statistical validation of a model using a serological approach. Vaccine 2003;21:32408. [35] Mahapatra M, Aggarwal N, Cox S, Statham RJ, Knowles NJ, Barnett PV, et al. Evaluation of a monoclonal antibody-based approach for the selection of foot-and-mouth disease (FMD) vaccine strains. Vet Microbiol 2008;126: 4050. [36] Crowther JR, McCullough KC, Brocchi E, De Simone F. Monoclonal antibodies against FMDV: use and potential application. In: Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of FMD. Rome: FAO; 1984. p. 40.

governmental decisions made in each country. The data collected in this work will hopefully help to contribute in this direction. Acknowledgements This work was supported by the Consejo Nacional de Investigaciones Cientcas y Tcnicas (CONICET) of Argentina, FONCYT (PID2003-00330) and the Animal Virology Studies Foundation (FEVAN). CEVAN and SENASA are members of the Inter-institutional FMD Research and Development Network (RIIDFA) of Argentina. We thank Miss Silvia Rojana, Maria Rodriguez and Carmen Devincenzo for their technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vaccine.2008.09.033. References
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