This document describes the development of a multiplex lateral flow device (LFD) for the on-field identification and serotyping of foot-and-mouth disease virus (FMDV) serotypes O, A, and Asia 1. The multiplex LFD utilizes the same monoclonal antibodies as a validated FMDV antigen detection and serotyping ELISA. It provides results in 15 minutes, identifying pan-FMDV positivity as well as the specific serotype. Testing showed it had diagnostic performance comparable to ELISA, correctly identifying field samples as types O or A. The multiplex LFD is well-suited for timely on-site FMD diagnosis and serotyping without equipment in endemic areas.

1. A MULTIPLEX LATERAL FLOW DEVICE FOR ON-FIELD IDENTIFICATION
AND SEROTYPING OF FOOT-AND-MOUTH DISEASE VIRUS
INTRODUCTION
1 Istituto Zooprofilattico
Sperimentale della
Lombardia e dell’Emilia
Romagna (IZSLER),
Brescia, Italy.
2Department of
Chemistry, University of
Turin,
Torino, Italy
3In3diagnostic, s.r.l.
Grugliasco (Torino), Italy
4Department of
Veterinary Science,
University of Turin,
Grugliasco (Torino), Italy)
GRAZIOLI Santina1
ANFOSSI Laura2
NAGAROL Chiara3
CAVALERA Simone2
FOGLIA Efrem A.1
PEZZONI Giulia1
ROSATI Sergio4
BROCCHI Emiliana 1
Corresponding authors:
Emiliana Brocchi
E-mail: emiliana.brocchi@izsler.it
emiliana.brocchi@gmail.com
Santina Grazioli
Santina.grazioli@izsler.it
CONCLUSION
MATERIAL AND METHODS
RESULTS
Acknowledgments
The study was funded by
the National Grant for
the research project
PRC 02/2020.
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 12Sample 11Sample 10Sample 9Sample 8Sample 7Sample 6
Analitical Sensitivity Limit of Detection range (TCID50)
FMDV type O (O Manisa and O Panasia 2) 6,3X102 ↔ 103
FMDV type A (A22 Iraq and A G-VII) 3,2X102 ↔ 1,5X103
FMDV type Asia1 (Shamir and Sind-08) 4,3X103 ↔ 3,2X104
In foot-and-mouth disease (FMD) endemic regions, which often lack adequate transport systems and equipped laboratories, Lateral Flow
Devices (LFD) represent the simplest tool for rapid on-site diagnosis. A commercially available pan-serotype LFD offers a user-friendly
disease confirmation tool, but lacks serotyping capacity, whilst serotype identification is crucial to identify regional transmission patterns
and to ensure appropriate vaccines selection in countries where multiple serotypes co-circulate.
This study describes the development of a multiplex LFD, based on well characterized monoclonal antibodies (MAbs), for FMD diagnosis and
simultaneous serotyping of FMDV O, A and Asia 1 in a single strip.
The novel multiplex LFD exploits the same set of serotype-specific capturing MAbs validated for the FMDV Antigen detection and serotyping
ELISA (1) and a pan-FMDV MAb linked to gold nanoparticles as signal reporter, enabling naked-eye detection after 15 minutes.
In a single strip, five reactive lines reveal respectively: pan-FMDV positivity (test line 1), positivity to type O, Asia 1 or A virus (test lines 2, 3
and 4), and test validity (control line).
Test optimization required formulation of an appropriate running buffer, selection of best reagents concentrations and best sequence position of the
multiplereactive lines in the single strip to prevent interferenceor competition between pan-FMDand type specific reactions.
FMDV reference strains and known epithelium homogenates from outbreaks occurred in Tanzania were used for an initial evaluation of the novel
multiplex LFDand for comparativeanalysis with Antigen-ELISA and real-timeRT-PCRs(pan-FMDVand topotype specific for pool 4 viruses).
For each serotype, analytical sensitivity was evaluated on two reference
viruses in parallel with Ag-ELISA. The reference strains were correctly
classified with analytical sensitivity similar to that shown by the Ag-
ELISA and absence of cross-reactivity between serotypes. The pan-
FMDV test line was clearly visible, irrespective the serotype analysed.
Diagnostic performance
Five field samples (epithelium homogenates) positivefor type O and nine positive for type A produced pan-FMDV and type-specific reactions coherent
with Ag-ELISAresults.
The performance of the Antigen-ELISA and the multiplex LFD was comparable as the same catching and detector monoclonal
antibodies are used in both kits. Thanks to its multiplexing capability and maximum simplicity, the LFD is suited for timely on-site
FMD diagnosis with simultaneous identification of three serotypes within minutes and without requiring any equipment.
Additional validation of the technology in endemic areas is planned.
Studies to develop a multiplex LFD for typing of SAT1 and SAT2 FMD viruses are in progress.
Sample pad
Probe pad
anti-FMDV-A
Adsorbent pad
Control
Test lines
anti-FMDV-Asia 1
anti-FMDV-O
TO
anti-mouse IgG
TATAsia1TPan
anti-FMDV Pan
Gold nanoparticle
labelled with Pan-FMDV MAb
Ctrl
TPan
TO
TA
TAsia1
Ctrl
TPan
TO
TA
TAsia1
NEG
Ctrl
TPan
TO
TA
TAsia1
Ctrl
TPan
TO
TA
TAsia1
FMDV-O FMDV-A FMDV-Asia1
Ctrl
TPan
TO
TA
TAsia1
FMDV-unknown
FMDVtype O samples FMDVtype A samples
FMDV Sample origin
Sample
ID
Ag-ELISA (OD) real time PCR (Ct) LFD
*Type-spMAb pan-1F10
pan-FMDV
(3D)
topotype-sp
(VP1)
pan-FMDV type-specific
TYPE O
O/TANZ Ring'wani18 1 0,9 2,9 7 7 + +
O/TANZ Mbilikili/2014 2 - - 17 18 - -
O TANZ Miseke/2014 3 1,8 3 14 15 +++ +++
O/TANZ Miseke/2014 4 1,2 1,8 12 13 +++ +++
O/TANZ Kemgesi/2018 5 0,8 2,9 15 16 +++ +++
TYPE A
A/TANZ Motuker/2015 6 2,9/3,2 3 13 19 ++ ++
A TANZ Kyabakari/2012 7 - - 20 21 - -
A/TANZ Natambiso/2015 8 3/2,9 3,2 9 7 ++++ +++
A/TANZ Park Nyigoti/2012 9 2,2/2,9 2,3 19 19 ++++ +++
A/TANZ Nyichoka/2015 10 0,8/1,7 0,5 13 13 + +++
A/TANZ Robanda/2015 11 0,4/0,9 0,3 22 23 + +++
A/TANZ Robanda/2015 12 1,3/2,2 1,7 15 16 +++ +++
A/TANZ Nyichoka/2015/1 13 2,4/3,1 1,9 15 14 +++ +++
A/TANZ Nyichoka/2015/3 14 2,7/3,3 2,9 14 14 +++ +++
* Two separate Mabs for type A; pool oftwo Mabs fortype O
typeC typeSAT1
Reference FMDV strains
The multiplex LFD was evaluated in parallel
with the Antigen and serotyping ELISA kit
References
Grazioli S, Ferris NP, Dho G,
Pezzoni G, Morris AS,
Mioulet V, Brocchi E.
Development and
validation of a simplified
serotyping ELISA based on
monoclonal antibodies for
the diagnosis of foot-and-
mouth disease virus
serotypes O, A, C and Asia
1. Transbound Emerg Dis.
2020 Jun 12. doi:
10.1111/tbed.13677