This study compared the diagnostic performance of three commercial ELISA kits for detecting antibodies against foot-and-mouth disease virus type O (FMDV-O) in cattle and pigs in South Korea. Sera from vaccinated and infected animals were tested using the three ELISA kits and results were compared to virus neutralization tests (VNT). The domestic ELISA kits generally showed higher sensitivity than the international kit, though the international kit had higher specificity. All three kits showed moderate to substantial agreement with VNT results. This evaluation of diagnostic performance can help ensure reliable serological surveillance for FMDV-O in South Korea.

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EuFMD | Open Session special edition | #OS20se
Comparison of Diagnostic Performances of Three Commercial ELISA Kits
for Detection of Antibodies to Foot-and-Mouth Disease Virus Type-O
Doheon Kwon, YouJin Han, JiHyun Lee, Hyun Mi Pyo, Eun-Jin Choi, Wonseok Shin, JaeMyoung Kim and Mi-Young Park
Foot and Mouth Disease Diagnostic Division, Animal and Plant Quarantine Agency, 177, Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do, Republic of Korea
INTRODUCTION
Vaccination has been the main strategy to prevent and control Foot-and-mouth Disease
(FMD) in Korea since 2010 and three FMD vaccines containing various strains has been
routinely used under national vaccination program since 2019. Furthermore, three
commercial ELISA kits have been licensed for detection of antibodies against structural
proteins (SP) of FMDV (Foot-and-mouth Disease Virus) to estimate vaccine-induced
immunity. These include PrioCHECK SP Type-O ELISA, accredited internationally and
two domestic solid-phase ELISA kits coated with recombinant protein-based antigens,
the latter two of which were approved in 2020. Since these various factors were
involved, variability in serological test results owing to differences in the antigenic
specificity of tests according to the particular virus strains and antibody reagents
utilized in the tests may affect the reliability of serological results. Therefore, we
investigated diagnostic performances of three ELISAs used for sero-surveillance to
monitor vaccine coverage and assess herd immunity in cattle and pigs.
MATERIALS & METHODS
A total of three FMDV SP type O ELISA kits licensed in Korea (Table 1) were evaluated
using 1540 sera collected from pigs and cattle vaccinated with one of three FMD
vaccines as described below (Table 2). In addition, 153 sera from unvaccinated pigs
experimentally infected with FMDV O/SKR/BE/2017 or O/SKR/AS/2019 and 368
negative sera from unvaccinated and uninfected animals were tested too. Also,
neutralizing antibody (NA) titers were determined with virus neutralization tests (VNT)
against homologous virus strains.
RESULTS
Country Supplier Serotype Antigens PD50
a
UK
Merial Animal
Health Ltd.
O+A
O Manisa, O 3039,
A22 Iraq
≥3PD50
Russia FGBI ARRIAH O+A
O Primorsky,
A Zabaikalsky
≥6PD50
Argentina Biogenesis Bago S.A O+A
O1 Campos, A24 Cruzeiro,
A2001 Argentina ≥6PD50
Table 2. Summary of FMD vaccines approved for routine use in Korea
Table 1. Characteristics of SP type-O ELISA kits tested
Test-kit
PrioCHECK FMDV
Type O ELISA (P)
Bionote FMD Type-O
Ab ELISA (B)
VDPro FMDV Type-O
Ab b-ELISA (M)
Country/Supplier
Netherlands/
ThermoFisher Scientific
Korea/Bionote
Korea/
Median Diagnostics
ELISA format Blocking ELISA Competitive ELISA Blocking ELISA
Antigen
Inactivated virus
(O1 Manisa, ME-SA)
coated with
trapping mAba
Recombinant VP4231
proteins (O/SKR/JC/2014,
SEA)
coated
Recombinant P13C
proteins (O/SKR/CJ/2000,
ME-SA) coated with
trapping mAb
Cut-off value* PIb≥50% PI≥50% S/Nc≤0.6
a Monoclonal antibody, b Percentage of inhibition, c Sample to negative ratio
*Interpretation of the result : positive (PI≥50%, S/N≤0.6) or negative (PI<50%, S/N>0.6)
Table 3. Sample description
a 50% Protective dose
Group Species
No. of
samples
Vaccine
(No. of samples)
Challenge virus
Vaccinated
Cattle 304 Ma (117), Ab (98), Bc (89) -
Pigs 1236 M (428), A (580), B (228) -
Infected Pigs
601) - O/SKR/AS/2019
932) - O/SKR/BE/2017
Negative3)
Cattle 213 - -
Pigs 155 - -
a Merial, b ARRIAH, c Bago S.A
1) Samples were collected daily from the day of challenge to 19 dpc (days post-contact).
2) Samples were collected daily from the day of challenge to 14 dpc, and after that every week until 162 dpc.
3) Bovine sera from Canada (n=113) and New Zealand (n=100), swine sera from Korea (n=3) and Canada (n=152)
Table 5. Detection rates of antibodies against SP of FMDV type-O in vaccinated animals
Species Group No. of samples
Detection rates of antibodies (%)
P B M VNT*
Cattle
M 117 86.3 100.0 97.4 89.7
A 98 99.0 100.0 99.0 100.0
B 89 95.5 98.9 96.6 97.8
Total : 304 93.1 99.7 97.7 95.4
Pigs
M 428 73.4 81.3 88.1 92.5
A 580 72.8 79.0 88.8 94.0
B 228 99.1 95.6 96.1 99.1
Total : 1236 77.8 82.8 89.9 94.4
The results of all test-kits demonstrated better detection rates of antibodies in sera from cattle vaccinated with
ARRIAH for bovine, whereas sera from pigs vaccinated with Bago resulted in higher detection rates of antibodies.
Also, two domestic kits yielded higher detection rates of antibodies than those of PrioCHECK kit except from pig sera
vaccinated with Bago. When compared to the results of VNT, ELISA kits tended to show lower detection rates of
antibodies than VNT in swine sera, while there was no significant difference between ELISA kits and VNT in bovine
sera.
* NA titer was measured against homologous strain used in vaccination (O Manisa for Merial vaccine, O Primorsky for
ARRIAH vaccine, O1 Campos for Bago vaccine). NA titers ≥16 were interpreted as positive.
Table 4. Diagnostic sensitivity and specificity of three ELISA kits relative to VNT
Species Group
DSea (%) DSpb (%) Kappa value3)
No. of
samples1) P B M
No. of
samples2) P B M P B M
Cattle Vc 290 95.2 100.0 98.3 227 96.9 90.3 93.0 0.918 0.913 0.917
Pigs
V 1167 81.4 83.7 91.3 204 96.6 84.3 81.9 0.547 0.516 0.646
Id 115 80.9 77.4 97.4 193 100.0 99.5 100.0 0.841 0.804 0.979
Total : 1282 81.4 83.2 91.8 242 97.1 86.8 84.7 0.565 0.534 0.687
Although specificities of PrioCHECK kit were higher than those of domestic kits, it was found that domestic kits
tended to show higher sensitivities, except the result of Bionote kit in the group of infected pigs. According to the
results of kappa values, all of three ELISA kits showed at least moderate agreement with VNT regardless of species
and vaccination against FMDV or infection with FMDV.
a Diagnostic sensitivity, b Diagnostic specificity, c Vaccinated, d Infected
1) Only samples showing positive results in VNT (VNT titers≥16) were selected for DSe.
2) Negative sera from unvaccinated and uninfected animals and samples from vaccinated or infected animals giving
negative results in VNT (VNT titers<16) were selected for DSp.
3) Moderate agreement: 0.401~0.600, substantial agreement: 0.601~0.800, almost perfect agreement: 0.801~1.000
DISCUSSION
The results of three ELISAs demonstrated lower positivity rates to SP antibodies
compared to those of VNT against homologous vaccine strains in sera from vaccinated
or infected animals with FMDV type-O. However, the sensitivities of three ELISAs are
supposed to be less affected by the specificity of antigens used in that the results of
kappa values between ELISAs and VNT were identified as moderate or higher level of
agreement. Also, detection rates of two domestic kits tended to be higher than those of
PrioCHECK kit from samples in vaccinated animals, which reflected that various vaccines
in use and field strains from outbreaks in Korea were considered in development of
ELISA kits.
REFERENCES
1. Li Y, et al. Evaluation of the solid phase competition ELISA for detecting antibodies against the
six foot-and-mouth disease virus non-O serotypes. J Virol Methods. 2012 Aug;183(2):125-31.
2. Chénard G, et al. A solid-phase blocking ELISA for detection of type O foot-and-mouth disease
virus antibodies suitable for mass serology. J Virol Methods. 2003 Jan;107(1):89-98.
3. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics.
1977 Mar;33(1):159-74.
Fig. 1. Kinetics of antibody responses to FMDV type-O in sera from pigs infected
with FMDV type-O using three ELISA kits and VNT.
The results were generated from serial samples (1~19 dpc) in pigs infected with FMDV O/SKR/AS/2019 (a), (b) and
from serial samples (1~162 dpc) of pigs infected with FMDV O/SKR/BE/2017 (c), (d). The red dotted line on each
graph indicates cut-off values. PrioCHECK kit and Median kit detected seroconversion as early as 5~7 dpc, which is
equivalent to the results of VNT (4~5 dpc) and Bionote kit detected seroconversion slightly later (7~10 dpc).
a days post-contact
* NA titer was measured against homologous strain used in vaccination. Antibody titers greater than 2.41 (log10256) were
considered as 2.71 (log10512) and antibody titers less than 1.20 (log1016) were considered as 1.00 (log1010).
Table 6. Detection rates of antibodies against SP of FMDV type-O in infected pigs
Challenge virus
No. of
samples
Detection rates of antibodies (%)
P B M VNT*
O/SKR/AS/2019 60 45.0 60.0 73.3 76.7
O/SKR/BE/2017 93 71.0 57.0 73.1 74.2
Total : 153 60.8 58.2 73.2 75.2
Median kit yielded higher detection rates of antibodies than other two kits did and its results were similar to those of
VNT.
* NA titer was measured against homologous strain used in challenge. NA titers ≥16 were interpreted as positive.
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
VNtiter(log10)
dpc (days post-contact)
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
1 2 3 4 5 6 7 8 9 10 14 21 28 49 56 70 162
VNtiter(log10)
dpc (days post-contact)
(a) O/SKR/AS/2019 (ELISA kits) (b) O/SKR/AS/2019 (VNT)
(c) O/SKR/BE/2017 (ELISA kits) (d) O/SKR/BE/2017 (VNT)
0
0.2
0.4
0.6
0.8
1
1.20
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
S/N(Sampletonegativeratio)
PI(Percentageofinhibition,%)
dpc (days post-contact)
P B M
0
0.2
0.4
0.6
0.8
1
1.20
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 14 21 28 49 56 70 162
S/N(Sampletonegativeratio)
PI(Percentageofinhibition,%)
dpc (days post-contact)
P B M