EuFMD OS20

1. 1
VALIDATION OF RECOMBINANT PROTEIN-BASED ELISA FOR DETECTION OF
ANTIBODIES TO FOOT-AND-MOUTH DISEASE VIRUS TYPE-O
Eun-Jin Choi1, YuJin Han1, Jieun Ryu2, Jungho Kim2, JiHyun Lee1, Hyun Mi Pyo1, WonSeok Shin1, DoHeon Gwon1, Jae Myung Kim1, Mi-Young Park1
1 Foot and Mouth Disease Diagnostic Division, Animal and Plant Quarantine Agency, Gimcheon, Gyeongsang buk-Do, Republic of Korea
2 BIONOTE Inc., 34, Samsung1ro 4-gil, Hwaseong-si, Gyeonggi-do, 18449, Republic of Korea
INTRODUCTION
Serological tests for detecting antibodies to structural proteins (SP) of Foot-and-
mouth disease virus (FMDV) are required to support assessment of vaccination
effectiveness and contribute to estimate the population immunity level for post-
vaccination monitoring. Although virus neutralization test (VNT) is prescribed in the
OIE-Manual as the golden standard method for determining SP antibodies to FMDV,
there may be disadvantages in terms of ease of use (high-level biocontainment
facilities, trained personnel, time-consuming, labor etc.) for massive screening
samples. Therefore, we have developed a new recombinant protein-based solid-
phase competitive ELISA (r-SPCE) with monoclonal antibody (Mab) for detection of
antibodies to FMDV type-O. This study describes the validation process in cattle and
pigs for this kit.
MATERIALS & METHODS
The r-SPCE kit was validated with field sera collected from either vaccinated or non-
vaccinated from non-infected cattle and pig farms, international reference sera
obtained from WRLFMD, Pirbright and sera from experimentally challenged animals.
RESULTS
The overall diagnostic specificity of 99.9% with a threshold of 50% inhibition was
identified by analyzing 1,070 cattle and 730 pigs from FMD-free and non-vaccinated
country (Canada, USA). Diagnostic sensitivity of 95.2% was estimated using 475
known positive animal sera. International reference sera were correctly detected. A
high level of serotype-specificity was demonstrated testing IAEA/FAO standard sera
strongly positive against other FMDV serotypes. Also, the coefficient of variation (CV)
obtained for evaluating reproducibility and repeatability estimated ≤1 0 %,
demonstrating the excellent stability of the r-SPCE.
Species No. of sample Specificity, % (No. of positives)
Bovine 1,070 99.0 (1)
Swine 1,170 100 (0)
Total 2,240 100% (1)
Table 1. Determination of cut-off (threshold) values
Table 4. Comparative evaluation of FMDV type-O ELISAs with VNT
Table 2. Result obtained with IAEA/FAO FMDV reference sera
Serotype Virus strain OD PI Result
O O1 Manisa 0.241 86.9 Pos.
A A 24 Cruzeiro 1.138 38.3 Neg.
Asia 1 Asia 1 Shamir 0.942 38.9 Neg.
SAT 1 SAT 1 1.273 30.9 Neg.
SAT 2 SAT 2 1.214 34.1 Neg.
SAT 3 SAT 3 1.594 13.5 Neg.
DISCUSSION
Establishing fully validated and reproducible methods for serological assessment of
detecting vaccine-induced antibodies needs considerable effort, particularly if a
various of vaccine and field strains are involved as in the case of Korea. The
recombinant protein-based SPCE developed for the detection of antibodies to
FMDV type-O demonstrated high specificity and sensitivity. In addition, this assay
offers advantages in terms of repeatability and broader cross-specificity, but also is
easy-to use and applicable to multiple species.
REFERENCES
1.Paiba GA, et al. J Virol Methods. 2004: 115(2):145-158.
2.Chénard G, et al. J Virol Methods. 2003: 107(1):89-98.
3.Kang Y, et al. BMC Vet. Research. 2018: 14:371.
Figure 1. Principle of solid-phase competitive ELISA using recombinant
VP4231 protein of FMDV O/JC/SKR/2014 (O/SEA/Mya98). ELISA plates
were coated with recombinant vp4231 antigen trapped by purified specific Mab. Test
samples and Mab-HRP conjugate are added to the wells of ELISA plate. During incubation,
if there are antibodies against O type in the test sample, the antibodies and HRP
conjugated Mabs of O type competitively bind to the antigens on the well. 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. The
enzyme activity will thus be directly inversely proportional to the anti-FMD SP antibodies
(serotype O) in samples. Colorimetric reading will be performed by using a
spectrophotometer at 450nm and reference wavelength at 620nm.
Table 3. Diagnostic specificity at the established cut-off
The optimal cut-off for percentage of inhibition (PI) at the dilution 1/5 was determined
using receiver operated characterstics (ROC) curves obtained by known negative and
positive sera with the VNT being the golden standard. Negative sera were originated
from 1,485 different animals in non-vaccinated and FMD-free area (Canada, USA).
Positive sera were from 475 vaccinated or infected animals, which shows positive result
in VNT (serum titers ≥1/16 were considered positive.). Fifty PI represented the
threshold that best discrimated negative and positive sera. Using this cut-off, the ELISA
scored specificity of 96.0%, sensitivity of 95.2% which resulted in the maximum
accuracy of 95.8%.
1
4
16
64
256
1024
0
10
20
30
40
50
60
70
80
90
100
110
120
DPI-0 DPI-3 DPI-6 DPI-10 DPI-17 DPI-24 DPI-31
VNTTITER
PIVALUE(%)
BIONOTE Prionics VNT
Figure 4. Comparison of FMDV-O r-SPCE and VNT using sera from cattle
infected with FMDV O/AD/SKR/2010. Samples from 5 cattles were collected
daily up to 10 days post infection (DPI), and then at 7 days intervals after 10 dpi. At 6
dpi all serum samples were positive for antibodies to FMDV-O, and remained positive
until the end of the experiments (31 dpi). When compared with gold standard test VNT,
same results were observed. For VNT, serum titers ≥1/16 were considered positive.
Species Status No. of
samples
Detection rate, % (Concordance, %)
VNT R-SPCE PrioCHECK
Bovine
Vaccinated 15 100 100 (100) 100
Infected 107 90.7 90.7 (100) 90.7
Sub-total 122 91.8 91.8 (100) 91.8
Swine
Vaccinated 440 74.1 82.3 (81.4) 76.6
Infected 18 88.9 88.9 (100) 100
Sub-total 458 82.5 77.5 (82.1) 75.3
Total 580 84.5 80.5 (85.9) 78.8
Figure 3. Distribution of the PI recorded from the examination of known
negative sera for cattle (n=1,070) and pigs (n=1,170).
Figure 2. ROC curve
analysis of results of
r-SPCE test. ROC analysis
of potential cutoff values for
serum samples using FMDV-
O r-SPCE kit. Calculated
values for sensitivity,
specificity and accuracy
considering VNT was set at
the threshold 50 PI.
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) was used to evaluate cross-reactivity generated by antibodies to heterologous
serotype in the FMDV-A rELISA kit. All sera were correctly identified.
FMDV-O r-SPCE test for negative sera from animals collected in FMD-free and unvaccinated
country was almost correctly identified showing only one false positive in 2,240 samples.
The sensitivity of the FMDV-O r-SPCE relative to the VNT was 80.5% (for cattle; 91.8%
and pigs; 77.5% respectively), and high agreement (concordance 85.9%) was observed
between two tests. Also, FMDV-O r-SPCE detected 82.9% SP antibodies to FMDV type-O
for clinical serum samples from domestic animals with vaccinations, exhibiting higher
sensitivity compared to 77.4% of PrioCHECK SP type-O ELISA.
No. of
samples
Cut-off (Percentage of Inhibition, PI Value)
≥ PI 30 ≥ PI 40 ≥ PI 50 ≥ PI 60 ≥ PI 70
Specificity, % 1,485 92.3 95.0 96.0 96.8 97.9
Sensitivity, % 475 98.7 97.5 95.2 90.3 81.5
Accuracy, % 1,960 93.9 95.6 95.8 95.3 93.9