1. SEROLOGICAL ELISAS BASED ON MONOCLONAL ANTIBODIES
AS DIAGNOSTIC TOOLS FOR LUMPY SKIN DISEASE
INTRODUCTION
a Istituto
Zooprofilattico
Sperimentale della
Lombardia e
dell’Emilia Romagna
(IZSLER),
Brescia, Italy
b Friedrich-Loeffler-
Institut (FLI), Federal
Research Institute for
Animal Health,
Insel Riems, Germany
Contacts:
Emiliana Brocchi
emiliana.brocchi@izsler.it
emiliana.brocchi@gmail.com
LSD is an OIE notifiable disease of cattle with potential for transboundary spread and relevant economic impact. The etiological agent
is the LSD virus (LSDV), genus Capripoxvirus, family Poxviridae. In 2015, LSD emerged in the EU causing outbreaks in Greece and then,
throughout 2016, in several Balkan countries. The disease is endemic in Africa and Middle East and is also reported from some West
Eurasian countries [1]. Since the lack of reliable and practical diagnostic tools for serological surveillance, the aim of our study was the
development of ELISA assays based on Monoclonal Antibodies (MAbs) for serological detection of LSD.
Both ELISAs consistently detected the seroconversion in all cattle by 14 days post infection (Fig.3). The cut-off values were
evaluated from results of 170 negative cattle
In infected goats, indirect-trapping ELISA clearly detected seroconversion in only three animals during the observation period,
resulting less sensitive (or less cross-reactive) than the competitive test, that revealed antibodies in all goats (Fig.4)
CONCLUSION
The assays developed are promising tools for the control of LSD diffusion, despite cross-reactivity due to common epitopes
between LSDV and GTPV
The availability of characterized MAbs may provide a strategic resource for further diagnostic tests and for improving knowledge on
Capripoxviruses antigenicity
MATERIAL AND METHODS
RESULTS
References:
1. Tuppurainen ESM, Venter EH, Shisler JL, Gari
G, Mekonnen GA, Juleff N, Lyons NA, De
Clercq K, Upton C, Bowden TR, Babiuk S,
Babiuk LA. Review: Capripoxvirus Diseases:
Current Status and Opportunities for Control.
Transbound Emerg Dis. 2017 Jun;64(3):729-
745. doi: 10.1111/tbed.12444. Epub 2015
Nov 13. PMID: 26564428; PMCID:
PMC5434826.
2. Möller J, Moritz T, Schlottau K, Krstevski K,
Hoffmann D, Beer M, Hoffmann B.
Experimental lumpy skin disease virus
infection of cattle: comparison of a field
strain and a vaccine strain. Arch Virol. 2019
Dec;164(12):2931-2941. doi:
10.1007/s00705-019-04411-w. Epub 2019
Sep 19. PMID: 31538254
Acnowlegments:
The work was funded
by National Grant for
the research project
PRC2016/001.
HRPO-conjugated
MAb
3H5 2C6 2F10 2F12
Catching
MAb
3H5 1,8 2 2,3 1,1
2C6 1,8 2 2,7 1,2
2F10 2,3 2,5 2 1,3
2F12 2 2,1 2,3 0,5
Fig. 1: WB against semi-purified LSD virus. (A) Reaction of two LSDV
specific MAbs (2F10 and 2C6), and a negative MAb. (B) Reaction of two
sera from LSDV infected cattle and a LSDV negative control serum.
ODvalue
0
0,25
0,5
0,75
1
1,25
1,5
1,75
2
1 2 3 4 5 6 7 8
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Percentageofcompetition
0
10
20
30
40
50
60
70
80
90
100
-2 0 7 14 21 28
0
dpi
7
10
14
21
28
23
ODvalue
-0,25
0
0,25
0,5
0,75
1
1,25
1,5
1,75
2
-2 0 7 14 21 28
Tab. 1 : Reactivity of the 4 MAbs used as catcher
and peroxidase-conjugates in sandwich ELISA.
Results expressed as optical density values
Fig. 4: Reactivity profile in competitive and trapping ELISA of sera collected sequentially from the experimentally infected goats
Days post infection Days post infection
Sequential sera from 8 goats
Monoclonal Antibodies (MAbs)
From a panel of 75 MAbs raised against LSDV, four recognizing a protein of 32-35 kDa
(Fig. 1A) were selected, purified and conjugated. Serum samples from LSDV infected
cattle recognised the same protein (Fig. 1B), which probably corresponds to the most
immunogenic protein of poxviruses. The ability of the 4 MAbs to reciprocally catch
and recognize inactivated LSDV from infected cultures was evaluated by sandwich
ELISA (Tab.1).
ELISA assays
Sandwich ELISA
Western Blotting
Two Mabs-based ELISAs have been developed
Competitive ELISA
Trapping-Indirect ELISA
LSD crude
virus
2F10 MAb
LSDV positive serum
LSDV negative
serum
Color development = POS
No color
HRPO Anti-
ruminant IgG MAb
HRPO Anti-
ruminant IgG MAb
No color = POS
LSDV positive
serum
LSD crude
virus
2F10 MAb
2C6 HRPO-MAb
Color development
LSDV negative
serum 2C6 HRPO-MAb
Percentageofcompetition
A B
14 Cattle 14 Cattle
Fig. 3: Seroconvertion detected in sera collected from 14 cattle experimentally infected and tested in competitive and trapping ELISA
LSDV positive
sera
35KDa 35KDa
8 Goats 8 Goats
Cut-off
Cut-off
Infected with LSDV-Neethling vaccine strain or an LSDV field isolate (cattle) and a goat poxvirus (goats)
Collected weekly up to 28 days after experimental infection.
70 sera from 14 cattle
28 sera from 8 goats
170 field negative cattle sera from some livestock in Northern Italy
Samples
LSDV positive
MAbs
Competitive ELISA Trapping-Indirect ELISA
BASELLI Stefano a,
SABINO Marcella a,
PEZZONI Giulia a,
GRAZIOLI Santina a,
HOFFMANN Bernd b,
WOLFF Janika b,
FOGLIA Efrem a,
ZANETTI Benedetta a,
BREGOLI Arianna a,
BROCCHI Emiliana a