1. The document describes the development of a solid-phase competitive ELISA for the detection of antibodies against Foot and Mouth Disease Virus type A. 2. The ELISA uses a recombinant P13C protein from FMDV type A as the antigen. Testing on cattle, pig, and goat samples demonstrated high specificity of 98.3% for cattle and 97.2% for pigs. 3. Validation against international reference sera and experimental samples showed excellent performance. The SPC-ELISA accurately identified all reference sera and had higher sensitivity than other tests for detecting antibodies in field samples.

1. 1
EuFMD | Open Session special edition | #OS20se
Development of solid-phase competitive ELISA for detection of type-A
Foot and mouth disease virus antibodies
YouJin Han1, JiHyun Lee1, Hyun Mi Pyo1, Sang Ho Jang2, BoKyu Kang2, Eun-Jin Choi1, Wonseok Shin1, Doheon Kwon1, JaeMyoung Kim1 and Mi-Young Park1
1Foot and Mouth Disease Diagnostic Division, Animal and Plant Quarantine Agency, 177, Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do, Republic of Korea
2MEDIAN Diagnostics Inc. 878, Sunhwan-daero, Dongnae-myeon, Chuncheon-si, Gangwon-do, 24399, Republic of Korea
INTRODUCTION
Foot-and-mouth disease (FMD) has been mainly controlled by prophylactic
vaccination and national FMD sero-surveillance in Korea. Serological tests for
detection of antibodies against virus structural proteins (SPs) are suited to
determine protective antibody responses induced by vaccination. In 2018,
Vaccine program has been changed to administer vaccine containing two
serotypes (O+A) for cows and pig routinely owing to the occurrence of Foot-
and-mouth disease virus (FMDV) type A from pigs in Korea. However, approx.
461,000 and 559,000 of serological samples were subjected on only FMDV
type-O SP antibody test. Although FMDV type-O Ab ELISAs currently in use for
the surveillance were approved for monitoring vaccine coverage, the
standardization of diagnosis for detection of antibodies to FMDV type-A has
been constantly in need to estimate vaccine efficacy and population immunity.
Therefore, a recombinant-protein based ELISA has been developed for the
measurement of antibodies to FMDV serotype A and has been validated using a
wide range of sera from cattle and pigs to assess the adoption for national FMD
sero-surveillance.
MATERIALS & METHODS
The new solid-phase blocking ELISA type-A (SPBE) for detection of SP
antibodies to FMDV type-A was validated using test panels of sera from naïve,
vaccinated, and from vaccinated-and sequentially infected animals, and
international reference sera FAO phase ⅩⅧ, IAEA FMD Control sera
RESULTS
Table 3. Result obtained with IAEA/FAO FMDV reference sera
Table 1. Determination of cut-off values for cattle and pigs
Species No. of samples
Specificity, %
(No. of positives)
Bovine 818 99.9 (1)
Swine 1,294 97.2 (27)
Goat 118 100 (0)
Total 2,230 98.3 (28)
Validation data show high specificity and excellent performance on reference
panels as well as on experimental or field sera. Therefore, the result of this
study indicates the SPC-ELISA is simple and robust serological test tool that
can replace the VNT for screening SP antibodies against FMDV type-A.
Fig. 1. Schematic diagram of FMDV A/SKR/2018 P13C gene cloning for
diagnostic antigen. The recombinant protein (rP13C) was derived from the P1 precursor
and 3C protease genes using a baculovirus-expressed system. The competitor monoclonal antibody
(mAb) for the ELISA was generated by immunizing a VP1 peptide corresponding to the GH loop for
high correlation of FMDV neutralizing antibodies.
Fig. 2. Principle of solid-phase blocking ELISA using recombinant
P13C protein of FMDV A/SKR/2018. ELISA plates were coated with rP13C antigen
trapped by purified specific mAb. Test samples and control are added to the wells of ELISA plate.
After incubation and washing, the conjugate is added. Anti-FMDV type A antibodies if it contains
in the test samples will bind to the FMDV type A protein and will block the binding epitope of
HRPO Anti-FMDV type A conjugated mAb. If binding of the HRPO conjugate is blocked by
structural protein antibodies in test samples, the unbound conjugate will be washed away and
less or no color will be developed.
Bovine
No. of
samples
Cut-off (S/N ratio)
0.5 0.6 0.7 0.8 0.9
Specificity, % 560 99.5 99.5 99.1 94.5 65.7
Sensitivity, % 301 92.7 97.7 98.7 99.7 99.7
Accuracy, % 861 97.1 98.8 99.0 96.3 77.6
ROC analysis of potential cutoff values for Bovine serum samples using FMDV SP-A ELISA
kit. Calculated values for sensitivity, specificity and accuracy considering VNT was set at
the threshold 0.7 for cattle.
* S/N : Sample to Negative, VNT pos. = ≥1/16
Swine
No. of
samples
Cut-off (S/N ratio)
0.6 0.7 0.8 0.9 1.0
Specificity, % 460 100.0 100.0 98.3 76.3 34.8
Sensitivity, % 1436 77.3 83.1 88.6 95.0 99.1
Accuracy, % 1896 82.8 87.2 90.9 90.5 83.5
ROC analysis of potential cutoff values for swine serum samples using FMDV SP-A ELISA
kit. Calculated values for sensitivity, specificity and accuracy considering VNT was set at
the threshold 0.8 for pigs.
* S/N : Sample to Negative, VNT pos. = ≥1/16
Fig. 3. Frequency distribution of S/N ratio for negative and positive sera
generated from same samples as table 1 and 2 (n=861 for cattle, 1,896
for pigs). The cut-off is set in the S/N % zone where there is minimal overlap between the
positive and negative area. In the above graphs, the cut-off is set at 0.7 for cattle and 0.8 for pigs.
Neg. Pos.
Neg. Pos.
Serum panel from the IAEA (through the Animal Production and Health sub-programme
of the Joint FAO/IAEA Division from infected cattle which includes 6 FMDV serotypes (A, O,
Asia 1, SAT 1, SAT 2 and SAT 3) and WRLFMD (World reference Laboratory for Foot-and-
Mouth Disease). All sera were correctly identified.
Table 5. Comparative evaluation of two FMDV type-A ELISAs with VNT
Result of experimental sera from vaccine potency tests were compared with those of VNT
obtained against homologous strains. For cattle, result of higher agreement with VNT was
identified than for pigs. VNT pos. = ≥1/16
FAO
Reference
Panel
(Bovine)
Virus strain Sample S/N Result
O Manisa weak pos. 0.93 Neg.
O Manisa strong pos. 1.02 Neg.
O SKR 1/2000 weak pos. 1 1.26 Neg.
O SKR 1/2000 strong pos. 1.16 Neg.
C Noville weak pos. 1 0.92 Neg.
C Noville strong pos. 1.10 Neg.
Asia1 Shamir weak pos. 1 0.95 Neg.
Asia1 Shamir strong pos. 0.92 Neg.
A IRN 1996 weak pos. 0.64 Pos.
A IRN 1996 strong pos. 0.62 Pos.
IAEA
FMD
Control
Sera
(Bovine)
Serotype S/N Result
A 0.63 Pos.
Asia 1 0.79 Neg.
SAT 1 0.98 Neg.
SAT 2 1.02 Neg.
SAT 3 0.99 Neg.
Table 4. Comparative diagnostic sensitivity of FMDV type-A ELISA with VNT
Bovine (317)
VNT N. 317 PV sera
Pos Neg Detection rate of ELISA = 96.8%
ELISA
Pos 297 10 Detection rate of VNT = 95.0%
Neg 4 6 Concordance = 95.6%
Swine (1,616)
VNT N. 922 PV sera, 694 Infection sera
Pos Neg Detection rate of ELISA = 82.5%
ELISA
Pos 1,272 62 Detection rate of VNT = 88.9%
Neg 164 118 Concordance = 86.0%
Species No. of
samples
Detection rate, % (Concordance, %)
FMDV type-A PrioCHECK VNT
Bovine 706 97.3 (96.6) 93.8 (93.9) 95.6
Swine 922 90.1 (89.3) 20.3 (27.5) 92.1
Goat 88 38.6 (90.9) 36.6 (84.1) 45.5
Total 1,716 90.9 (92.4) 51.3 (57.8) 91.1
Table 6. Result of diagnostic specificity for FMDV type-A ELISA kit
The sensitivity of the FMDV Type-A ELISA and PrioCHECK relative to the VNT was 92.4%
and 57.8% respectively. Also, SPC-ELISA detected 90.9% SP antibodies to FMDV type-A for
clinical serum samples from domestic animals with vaccinations, exhibiting higher
sensitivity compared to 51.3% of PrioCHECK SP Type-A ELISA.
Specificity was assessed with naïve sera from FMD free area and non-vaccinated areas
(CANADA and USA). Specificity for naïve animals was very high on both bovine and
swine, and particularly on goat.
Conclusions
Table 2. Determination of cut-off values for pigs