LSD symposium - G. Pezzoni - Development of a pen-side test for the detection of LSDV based on monoclonal antibodies

1. DEVELOPMENT OF A PEN-SIDE TEST FOR
THE DETECTION OF LSDV BASED ON
MONOCLONAL ANTIBODIES
LSD Symposium - How Science can Support Disease
Management and Control, Rome 14-16 March 2023
Pezzoni Giulia
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna (IZSLER)

2. LSD Symposium - How Science can Support Disease Management and Control, Rome 14-16 March 2023
Introduction-Capripoxviruses
• dsDNA virus 170-250kb
• Family: Poxviridae
Lumpy skin disease virus (LSDV) Sheep pox virus (SPPV) Goat pox virus (GTPV)

3. LSD Symposium - How Science can Support Disease Management and Control, Rome 14-16 March 2023
Lumpy Skin disease 2015-2022
Sheep and Goat pox 2015-2022
Introduction-Capripoxviruses
Virus detection relies mainly on molecular
tests performed in the laboratory
https://wahis.woah.org/#/dashboards/country-or-disease-dashboard
Reliable and easy-to-use point-of-care devices
can be helpful in the early detection and
control of Capripoxvirus diseases
Especially where the absence of adequately
equipped laboratories makes difficult their
control

4. LSD Symposium - How Science can Support Disease Management and Control, Rome 14-16 March 2023
Summary
✓ Describe the development of a Lateral Flow Device (LFD) for LSDV virus detection using
two characterized LSDV-specific monoclonal antibodies (MAbs)
✓ Present the preliminary results on the evaluation of the LFD on Lumpy skin disease by
measuring its analytical and diagnostic performances
✓ Evaluation of the LFD on other Capripoxvirus

5. LSD Symposium - How Science can Support Disease Management and Control, Rome 14-16 March 2023
Sandwich ELISA
• 2F12 mAb
Capture
Detector
• 3H5, 2C6 and 2F10 mAbs
Capture
Detector
MAbs characterization by sandwich and competitive ELISA
Competitive ELISA
✓ Seventy-five hybridomas tissue cultures producing monoclonal antibodies (mAbs) were obtained against the LSDV Neethling strain
✓ Fourteen MAbs were mapped against the structural protein P32, within them, three were selected namely 3H5,2C6 and 2F10

6. LSD Symposium - How Science can Support Disease Management and Control, Rome 14-16 March 2023
LATERAL FLOW DEVICE DEVELOPMENT
• LFDs were tested both in single epitope (SE) and in double
epitope (DE).
• Different sizes of AuNPs were evaluated to identify the most
suitable for the test.
• The most efficient LFD was selected as a double epitope
(2C6 and 2F10_AuNPs).
LSDV antigen #2F10_AuNP #2F10 #2C6
Capture mAb
Detection mAb
Capture mAb
Streptococcal G
protein
epitope 1
epitope 2
T
C
T
C
C
T
POS
NEG Invalid

7. LSD Symposium - How Science can Support Disease Management and Control, Rome 14-16 March 2023
Analitycal sensitivity and specificity
0
0.5
1
1.5
2
2.5
TQ 1/3 1/9 1/27 1/81 1/243 1/729
LSDV dilution
OD
neat
▪ LFD detected LSDV up to ̴103,4 TCID50/ml
LSDV dilution in sample buffer
Bovine papular stomatitis virus
(1993,1998,2008,2020 isolates)
Pseudocowpox virus
(2009 and 2020 isolates )
▪ LFD showed 100% specificity
▪ Sandwich ELISA detected LSDV up to ̴103,6 TCID50/ml.
Orf viruses and Lentivirus

8. LSD Symposium - How Science can Support Disease Management and Control, Rome 14-16 March 2023
LFD- Lumpy Skin Disease Virus Detection
0
0.5
1
1.5
2
2.5
3
3.5
10 15 20 25 30
Evaluation of 20 skin nodules, from two different experimental infections
qPCR Ct values
LFD
signal
intensity
11 samples were collected 9 days post-infection (DPI) (LSDV Macedonia-2016)
9 samples were collected at various DPI (LSD Albania-2016)
✓ All the samples were positive in qPCR
✓ A total of 16 out of 20 skin nodules resulted in a positive
signal in LFD
✓ qPCR positive samples with Ct values higher than 24 can
result in weak positive or negative in LFD (borderline)
✓ qPCR positive samples with low Ct values (<20), can show a
weak signal in LFD. Presence of blood in the sample from
animals with the humoral response?
Correlation between LFD signal intensity and qPCR Ct values
+++/3 ++/2 +/1 -/0
C
T
qPCR according to Bowden et al., 2008 and Wolff et al. 2021

9. LSD Symposium - How Science can Support Disease Management and Control, Rome 14-16 March 2023
LFD- Capripoxviruses
Isolate qPCR
(Ct 1/2)
1/2 1/4 1/8
SPPV
SPPV-UAE_V330
21,6 + + +/-
SPPV-Egypt_V293
21,9 +/- + +/-
SPPV-India_V123
21,2 +/- + +/-
SPPV-Russia_V104
20,7 + +/- -
GTPV
GTPV-India_V103
21,9 +/- + +/-
LSDV
LSDV-
Bangladesh2021_V
393
20,8 ++ + +/-
Evaluation Capripoxviruses isolates
Dilution
✓ Both SPPV and GTPV have been detected
✓ The LSDV strain with a similar genome load with
SPPV and GTPV resulted in a higher LFD signal
✓ A reduced analytical sensitivity of the LFD for
SPPV and GTPV may be related to the presence of
different infectious virus particles?
P32
qPCR according to Bowden et al., 2008 and Wolff et al. 2021

10. LSD Symposium - How Science can Support Disease Management and Control, Rome 14-16 March 2023
LFD- Sheep pox virus
Evaluation of six skin nodules,
from naturally infected sheep
LFD - - +++ +++ + + +++ +++
qPCR 17,7 13,7 15,2 12,7 11,5
DUPLICATE DUPLICATE DUPLICATE
LFD + ++ ++ + + + +/-
qPCR 14,7 13 14 16 16,9 17 18,3
Tq 1/2 1/4 1/8 1/16 1/32 1/64
C
T
C
T
✓ All the samples were positive in qPCR
✓ A total of 5 out of 6 skin nodules resulted in a
positive signal in LFD
✓ A strong antibody level by double antigen ELISA
(IDVet) was present in the animal with a negative
result. If blood is present in the sample, it is likely
that the serum antibodies hamper the antigen
capture by LFD Mabs
qPCR according to Bowden et al., 2008

11. LSD Symposium - How Science can Support Disease Management and Control, Rome 14-16 March 2023
LFD- Oral Swab
✓ Tested a total of 10 oral swabs from LSDV
experimentally infected cattle
✓ All the samples were positive in qPCR with Ct
values ranging from 23,5 to 30,5
✓ A total of 4 out of 10 resulted in a positive signal
in LFD with a weak signal
✓ Tested a total of 15 oral swabs from field sheep
✓ All the samples were positive in qPCR with Ct values
ranging from 23,9 to 34,9
✓ All the samples resulted negative with LFD
LFD +/- + +/- +
qPCR 23,5 24,48 25 28,15
qPCR according to Bowden et al., 2008 and Wolff et al. 2021

12. LSD Symposium - How Science can Support Disease Management and Control, Rome 14-16 March 2023
Conclusion
✓ LFD showed a good performance on Skin lesions from LSDV experimentally infected
cattle. More samples have to be tested to properly evaluate the diagnostic sensitivity
of the device.
✓ Oral Swabs showed not to be the ideal sample to be tested with LFD
✓ LFD designed with LSD-specific MAbs can be potentially used also for SPPV and GTPV
detection

13. Protecting people, animals, and the environment every day
Drawings: FAO/Chiara Caproni
Giulia Pezzoni,
Stefano Baselli
Santina Grazioli
Emiliana Brocchi
Federica Monaco
Laura Anfossi,
Simone Cavalera
Chiara Nogarol
Sergio Rosati
Bernd Hoffmann
Laboratorio Central Veterinario-Sanidad Animal,
Algete, Spain. Montserrat Agüero
Thank you
Project founded by EuFMD-FAR – 2022