The European Commission for the Control of Foot-and-Mouth Disease (EuFMD), one of FAO’s oldest Commissions, came into being on the 12th June 1954, with the pledge of the sixth founding member state to the principles of a coordinated and common action against Foot-and-mouth Disease.
Results from a Interlaboratory exercise to evaluate NSP kits (C. Browning)
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
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
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
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)