EuFMD OS20

1. DEVELOPMENT OF A LIQUID-PHASE BLOCKING ELISA BASED ON FOOT-
AND-MOUTH DISEASE VIRUS A/YEONCHEON/2017 FOR POST-
VACCINATION SERO-MONITORING
J. Choi1, S. Jung1, S. Lee1, H-H. Kim1, J-H. Park1, and J. Kim*1
1Center for FMD Vaccine Research, Animal and Plant Quarantine Agency, Gimcheon, Korea
LPBE
Positive Negative Total
specificity
(%)
sensitivity
(%)
VNT
Positive 77a
12b
89
93.2*
86.7**
Negative 3c
41d
44
Introduction
A liquid-phase blocking (LPB)-ELISA has been
standardized to be used for serological
assessment of herd immunity against foot-and-
mouth disease virus (FMDV) because this assay is
the relatively simple and high-throughput
procedure compared to virus-neutralization test
(VNT). In this study, we developed LPB-ELISA to
measure the level of antibodies against serotype A
FMDV and validate its performance using an
extensive range of sera from pigs. The developed
LPB-ELISA used inactivated purified 146S particles
of FMDV A/Yeoncheon/SKR/2017 (A/Asia/Sea-
97/G2 lineage) which has been developed for
possible future use.
Materials and methods
FMDV A/Yeoncheon strain (A-YC) was cultivated
in BHK21 cells and then, viral activity was
chemically inactivated by BEI. The 146S particles
of the inactivated A-YC were purified by sucrose
gradient ultracentrifugation, and then used for an
ELISA antigen. Two polyclonal hyperimmune sera
against A-YC, one for capture antibody (rabbit)
and the other for detector antibody (guinea pig)
were prepared and optimized for LPB-ELISA
reaction. After optimization of ELISA reaction, the
developed LPB-ELISA was applied for antibody
titration against A-YC using the field-collected pig
sera.
Results
To evaluate the diagnostic performance of the
developed LPB-ELISA, a total of 133 sera obtained
from pigs vaccinated with serotype A monovalent
vaccine (experimental vaccine incorporated FMDV
A-YC) were screened in parallel by the virus
neutralization test (VNT). When test sera at a 1:10
dilution with a cut-off point of 50% inhibition of
reaction, LPBE exhibited proper analytic
performance with a high diagnostic sensitivity
(92.1%) and specificity (88.6%) in comparison with
the VNT (Table 1). In addition, the developed LPBE
has a high correlation with the VNT (r2 =0.724)
(Fig. 2).
Optimization of LPB-ELISA reactions
A
B
C
Fig. 2 On the basis of the checkerboard titration,
optimum ELISA reactions were determined. In
panel, A indicates the reactivity of antigen(A-YC)
with homologous pig sera in a sandwich ELISA after
serial dilution. B and C show the results of titration
using polyclonal rabbit (capture) and guinea pig
(detector) antibody, respectively.
Correlation between VNT and LPB-ELISA
Table 1. Specificity and sensitivity of LPB-ELISA for
the determine of FMDV antibodies in pigs received
with monovalent FMD A-YC vaccine. * Specificity
= (a/a+b) x 100, ** Sensitivity = (d/c+d) x 100
Fig. 2 Correlation and linear regression of log 10
antibody titers obtained by LPB-ELISA and by VNT.
Discussion
Antigenic variants continue to emerge within
each serotypes of FMDV, causing a major problem
in FMD diagnosis. Because the currently approved
and used FMD vaccines in Republic of Korea
consist of varying formations of inactivated
viruses of different serotypes, a more precise
diagnosis is required to detect FMDV specific
antibodies raised against vaccination.
In Republic of Korea, the SP-ELISA is adopted as
a screening method for evaluating herd immune
status after FMD vaccination because VNT has
certain practical limitation. However, whether
type A SP-ELISA can ensure the vaccination status
of individual animals and herds is unclear due to
antigenic variability among FMDV type A. In this
study, we developed the LPB-ELISA by using 146S
antigens of FMDV A-YC. In comparative analysis,
the developed LPB-ELISA showed considerably
higher sensitivity than SP-ELISA. The high false
negative rate of SP-ELISA demonstrated that the
test method may not guarantee a true vaccination
coverage. Therefore it can be concluded that the
LPBE is more suitable than SP-ELISA for use as a
screening test for the detection of antibodies
against FMDV type A.
Reference
[1] Basagoudanavar SH et al., 2013. Arch Virol.
158, 993-1001.
Fundamental principle of LPE-ELISA
In comparative evaluation of the ELISA tests, the
LPBE exhibited a better diagnostic performance
than SP-ELISA. The sensitivity of LPBE (86.5%) was
considerably higher than that of SP-ELISA (15.7%),
while the specificity of LPBE (93.2%) was almost
equivalent to that of SP-ELISA (100%) (Table 1 and
2.)
Correlation between VNT and SP-ELISA
SP-ELISA
Positive Negative
specificity
(%)
sensitivity
(%)
VNT
Positive 14a
75b
15.7*
100**
Negative 0c
44d
Table 2. Specificity and sensitivity of SP-ELISA for
the determine of FMDV antibodies in pigs
received with monovalent FMD A-YC vaccine. *
Specificity = (a/a+b) x 100, ** Sensitivity = (d/c+d)
x 100