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

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

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 virusE. 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

Method

NSP Harmonisation Study Plan

n=60

n=60

n=60

SamplesPositive

90 pos

90 posNegative

1800 x of serum

PrioCHECK

K

ID.Vet (short) ID.Vet (long)

Operator

K

In duplicate

K

360 aliquots of serum per

Laboratory

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

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.V

et

Sh

ort

NS

P E

LIS

A

PrioCHECK NSP ELISA

-/+

-/- +/-

+/+

Concordance

95.5%

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

0

10

20

30

40

50

60

70

80

90

100

C16-1093 C16-1106 C16-1099 S10-1052 S09-1393 S09-1040

Pe

rcen

tage

In

hib

ibtio

n

False Positive Sample Aliquots

Heat Inactivation of NSP

False Positive Samples

Titration Curves

0

10

20

30

40

50

60

70

80

90

100

1/5 1/10 1/20 1/40 1/80

Pe

rcen

tage

In

hib

itio

n

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

Per

cen

tage

Inh

ibit

ion

Titration Curves of 6 False Positive Samples

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

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

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)