OS18 - Open Session 2018 Borgo Egnazia (Puglia), Italy 29 - 31 /10/2018 EuFMD Sessions\Open Session\Archive-2018\Open 2018 - Italy\6. Online & Streaming\PPTs

1. Results from a Inter-
laboratory exercise to
evaluate NSP kits
Clare Browning, Lissie Henry, Ginette Wilsden
Satya Parida, Lynne Hendry,
Tim Pollard, Anna Ludi, Donald King

2. Purpose of Study:
Looking at the concordance of 2 commercially available ELISA kits for
the detection of Non-structural protein antibodies to Foot and Mouth
Disease Virus.
1) ID.Vet
2) PrioCHECK
Proposed Study
PrioCHECK® FMDV NS Antibody ELISA

3. Update to the previous study
Vaccine 24 (2006) 6966–6979
Comparative evaluation of six ELISAs for the detection of antibodies to
the non-structural proteins of foot-and-mouth disease virus
E. Brocchi a,∗, I.E. Bergmannb, A. Dekker c, D.J. Paton d, D.J. Sammine, M. Greiner f,
S. Grazioli a, F. De Simone a, H. Yadin g, B. Haas h, N. Bulut i, V. Malirat b, E. Neitzert b,
N. Goris j, S. Parida d, K. Sørensen k, K. De Clercq j
Abstract
To validate the use of serology in substantiating freedom from infection after foot-and-mouth disease (FMD) outbreaks have been controlled
by measures that include vaccination, 3551 sera were tested with six assays that detect antibodies to the non-structural proteins of FMD virus.
The sera came from na¨ıve, vaccinated, infected and vaccinated-and-infected animals; two-thirds from cattle, the remainder from sheep and
pigs. The assays were covariant for sensitivity, but not necessarily for specificity. A commercial kit from Cedi-diagnostics and an in-house
assay from IZS-Brescia were comparable to the NCPanaftosa-screening index method described in the Diagnostic Manual of the World
Animal Health Organisation. Using these three tests the specificity and sensitivity for the detection of carriers in vaccinated cattle approaches
or exceeds 99% and 90%, respectively.
© 2006 Elsevier Ltd. All rights reserved.
Keywords: Foot-and-mouth disease; Non-structural proteins ELISAs; Tests performances

4. Method

5. NSP Harmonisation Study Plan
n=60
n=60
n=60
Samples
Positive
90 pos
90 posNegative
1800x of serum
PrioCHECK
K
ID.Vet (short) ID.Vet (long)
Operator
K
In duplicate
K
360 aliquots of serum per
Laboratory

6. Positive Serum
• Experimental serum (sampled at a min of 8dpi):
vaccinated and infected; non-vaccinated and infected
• Inactivation process
- 2 x BEI inactivated & Innocuity tested
Negative Serum
• Negative field sera from FMDV free countries
Definition of Sample type

7. 0
10
20
30
40
50
60
70
80
90
100
110
120
130
-30 -20 -10 0 10 20 30 40 50 60 70 80 90 100
ID.VetShortNSPELISA
PrioCHECK NSP ELISA
-/+
-/- +/-
+/+
Concordance
95.5%

8. Can we reproduce the false positive results?
Experiments:
1. Heat Inactivation at 56oC
for 30 minutes
2. Titration Curves
3. Replicate 72hrs at RT
Samples:
1. Aliquot from exercise Op 1
2. Aliquot from exercise Op 2
3. Original inside stock
4. Original outside stock

9. 0
10
20
30
40
50
60
70
80
90
100
C16-1093 C16-1106 C16-1099 S10-1052 S09-1393 S09-1040
PercentageInhibibtion
False Positive Sample Aliquots
Heat Inactivation of NSP
False Positive Samples

10. Titration Curves
0
10
20
30
40
50
60
70
80
90
100
1/5 1/10 1/20 1/40 1/80
PercentageInhibition
Dilution Series
Titration Curves of Positive Samples
Titration curves of NSP
positive samples as opposed
to the false positive samples.
0
10
20
30
40
50
60
70
80
90
100
1/5 1/10 1/20 1/40 1/80
Dilution Series
PercentageInhibition
Titration Curves of 6 False Positive Samples

11. Can you replicate negatives
becoming positive?
A fresh aliquot of each of the samples were used;
• aliquot left at room temperature 72 hours.
• Aliquot left in the fridge for 72 hours .
Sample ID
Percentage Inhibition
(Original)
Percentage Inhibition (RT
72hrs)
C16-1093 30 36
C16-1106 21 22
C16-1099 23 26
S10-1052 22 97
S09-1393 36 39
S09-1040 26 38
A10 Holland 83 82
VR04 91 91
VS78 69 69

12. The objectives of the study;
• Data supports the idea that these 2 commercial assays have equivalent
performance for the detection of FMDV NSP-specific Abs.
• Provide concordance and validation data to accredit alternative assays for
routine diagnostic purposes.
• Hopefully mitigating any potential supply difficulties that may arise during an
outbreak.
Study also highlighted: Storage & heat inactivation of serum samples;
• Heat inactivation plays an important role in reducing the non-specific binding
resulting in spurious results. It is not an isolated incidence, it has been seen
by other laboratories.
• Outbreak situation samples will be fresh and are tested quickly.
• Post vaccination studies however, longer periods of storage before shipment
and testing.
Conclusion

13. Thank you
To all the participants from the EU-RL
• France: Anses-Laboratooire Sante Animale
• Italy: Isituto Zooprofilattico Sperimentale della
Lombardia e dell’ Emilia Romagna (IZSLER)
• Netherlands: Wageningen Bioveterinary research
(WBVR)
• UK: Animal Plant & Health Agency (APHA)
• UK: The Pirbright Institute (TPI)