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Development of a fluorescent RBL reporter
system for diagnosis of porcine cysticercosis
Md. Shahadat Hossain1,2
, Philip Toye2
, Lian Thomas2,3
, Franco H. Falcone1
1
Justus Liebig University Giessen, 2
International Livestock Research Institute (ILRI), 3
University of Liverpool
Created with BioRender Poster Builder
Background & Objectives
→Porcine cysticercosis is caused by a zoonotic
neglected tropical disease parasite, Taenia solium in pig.
→PCC reduces pork value, affects food security and
livelihood of pig farmers.
→Tongue palpation and meat inspection are most widely
used diagnostic methods.
→Serological diagnosis is based on IgG, characterized
by low sensitivity.
→IgE plays the central role in metazoan parasitic
infections.
⊕ Objective 1: Development and characterization of
porcinized IgE reporter cell lines which can bind pig IgE.
⊕ Objective 2: Selection, cloning, and recombinant
expression of candidate allergens of T. solium, followed
by their validation as diagnostic antigens.
Test Principle
⊕ Porcinized IgE reporter system created using Rat
basophil leukaemia (RBL) cells stably transfected with
neuropeptide Y monomeric red fluorescent protein fusion
(RBL NPY-mRFP), located in granules (Fig.1).
α α
α
Rat IgE
β γ
Pig IgE Pig IgE
γ
β γ γ
β γ
γ
C
h
i
m
e
r
i
c
P
i
g
/
R
a
t
F
c
ε
R
I
α
P
i
g
F
c
ε
R
I
α
R
a
t
F
c
ε
R
I
α
Fig. 1 Creation of chimeric pig/rat FcεRIα cell line
⊕ Porcinized reporter system incubated overnight with
pig-IgE followed by stimulation with allergens, results in
IgE crosslinking, by allergens, followed by degranulation
of reporter cells (Fig.2).
β
γ
γ
α
β
γ γ
α β
γ
γ
α
β
γ γ
α
Allergens
IgE
FcεR1
IgE
RBL NPYmRFP-pig/rat FCεRIα
reporter system
Measures red florescent protein (mRFP), released by
stimulated cells in supernatant after 45 minutes of
incubation (Ex/Em 530/590 nm)
Fig.2 Activation of the porcinized IgE reporter system by IgE-
allergen interaction. Crosslinking of receptor-bound IgE by
allergen induces degranulation and mRFP release into
supernatant.
Methods
Selection of putative IgE-binding
antigens expressed by tissue
migrating oncosphere stage for
cloning followed by sequence
verification of successful clones;
allergenicity prediction of
diagnostic allergens.
Published proteomic
and transcriptomic data
A A T
G C G
T C
T G
Sequence analysis
Protein motifs (e.g. EF-
Hand, potease signatures)
Allergen Databases
(e.g. AllFam, Allergome,
AlgPred 2.0)
Tyagi et al. predicted
helminth 'allergens'
Nucleofection of NPY-mRFP
reporter with pig high affinity
IgE receptor (alpha chain)
Antibiotic selection of
stable transfectant
Clonal selection of best
porcine IgE reporters
Suitability as diagnostic antigens
(Specificity, Sensitivity, ROC curves)
T A A
T
C C
A T T
A
G G
T
A
C
G
C
G
A
T
C
G
T
A pTT
Allergens
Codon
optimization
Cloning
HEK-293 6E
cell culture
Transfection
Shaking
Incubator
Recombinant expression of
allergens in HEK293-6E cells
followed by chromatographic
purification
Results
⊕ Five candidate diagnostic T. solium
oncospheral allergens identified through
bioinformatics analysis (Fig.3) and
allergenicity confirmed by AlgPred 2.0.
Fig. 3 Candidate allergens of T. solium
⊕ Three HEK293-6E cell supernatants
transfected T. solium allergens showed
expected protein band size in Western blot
analysis (Fig.4).
⊕ K0A0S9: 19.8 kDa , Q2XNL7: 20 kDa,
and W8P1J2: 22.3 kDa.
10
17
26
34
43
55
72
kDa 1 2 3 4 5 6 7
19.8 kDa 20 kDa
22.3 kDa
Fig. 4 Successful protein expression of
transfected allergens in Western blot. Lane 1:
protein ladder; Lane 2: negative control, Lane
3-7: transfected T. solium allergens .
Conclusion
✓ Stably transfected chimeric pig/rat
FcεRIα reporter system has been created
(Fig.5)
✓ Successful cloning and sequence
verification of four recombinant T. solium
allergens in expression vector
✓ Successful recombinant expression
Fig. 5 Stable transfection and PCR confirmation
of chimeric pig/rat FcεRIα cell line. Fluorescence
microscopy showing red fluorescence from
transfected cells. In PCR, RBL-2H3 cells used
for negative control (SS-nc). SS: chimeric pig/
rat FcεRIα, MW is a 100 bp ladder.
Outlook
● Further expression and purification
of candidate diagnostic allergens.
● Validation and assessment of
Porcinized RBL reporter system
with diagnostic allergens.
● Screening of infected and non-
infected pig sera with reporter
system.
● Determination of specificity and
sensitivity.
Commissioned by the German Federal Ministry for Economic Cooperation and Development
(BMZ) and carried out by ATSAF e.V. on behalf of the Deutsche Gesellschaft für
Internationale Zusammenarbeit (GIZ) GmbH.

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Development of a fluorescent RBL reporter system for diagnosis of porcine cysticercosis

  • 1. Development of a fluorescent RBL reporter system for diagnosis of porcine cysticercosis Md. Shahadat Hossain1,2 , Philip Toye2 , Lian Thomas2,3 , Franco H. Falcone1 1 Justus Liebig University Giessen, 2 International Livestock Research Institute (ILRI), 3 University of Liverpool Created with BioRender Poster Builder Background & Objectives →Porcine cysticercosis is caused by a zoonotic neglected tropical disease parasite, Taenia solium in pig. →PCC reduces pork value, affects food security and livelihood of pig farmers. →Tongue palpation and meat inspection are most widely used diagnostic methods. →Serological diagnosis is based on IgG, characterized by low sensitivity. →IgE plays the central role in metazoan parasitic infections. ⊕ Objective 1: Development and characterization of porcinized IgE reporter cell lines which can bind pig IgE. ⊕ Objective 2: Selection, cloning, and recombinant expression of candidate allergens of T. solium, followed by their validation as diagnostic antigens. Test Principle ⊕ Porcinized IgE reporter system created using Rat basophil leukaemia (RBL) cells stably transfected with neuropeptide Y monomeric red fluorescent protein fusion (RBL NPY-mRFP), located in granules (Fig.1). α α α Rat IgE β γ Pig IgE Pig IgE γ β γ γ β γ γ C h i m e r i c P i g / R a t F c ε R I α P i g F c ε R I α R a t F c ε R I α Fig. 1 Creation of chimeric pig/rat FcεRIα cell line ⊕ Porcinized reporter system incubated overnight with pig-IgE followed by stimulation with allergens, results in IgE crosslinking, by allergens, followed by degranulation of reporter cells (Fig.2). β γ γ α β γ γ α β γ γ α β γ γ α Allergens IgE FcεR1 IgE RBL NPYmRFP-pig/rat FCεRIα reporter system Measures red florescent protein (mRFP), released by stimulated cells in supernatant after 45 minutes of incubation (Ex/Em 530/590 nm) Fig.2 Activation of the porcinized IgE reporter system by IgE- allergen interaction. Crosslinking of receptor-bound IgE by allergen induces degranulation and mRFP release into supernatant. Methods Selection of putative IgE-binding antigens expressed by tissue migrating oncosphere stage for cloning followed by sequence verification of successful clones; allergenicity prediction of diagnostic allergens. Published proteomic and transcriptomic data A A T G C G T C T G Sequence analysis Protein motifs (e.g. EF- Hand, potease signatures) Allergen Databases (e.g. AllFam, Allergome, AlgPred 2.0) Tyagi et al. predicted helminth 'allergens' Nucleofection of NPY-mRFP reporter with pig high affinity IgE receptor (alpha chain) Antibiotic selection of stable transfectant Clonal selection of best porcine IgE reporters Suitability as diagnostic antigens (Specificity, Sensitivity, ROC curves) T A A T C C A T T A G G T A C G C G A T C G T A pTT Allergens Codon optimization Cloning HEK-293 6E cell culture Transfection Shaking Incubator Recombinant expression of allergens in HEK293-6E cells followed by chromatographic purification Results ⊕ Five candidate diagnostic T. solium oncospheral allergens identified through bioinformatics analysis (Fig.3) and allergenicity confirmed by AlgPred 2.0. Fig. 3 Candidate allergens of T. solium ⊕ Three HEK293-6E cell supernatants transfected T. solium allergens showed expected protein band size in Western blot analysis (Fig.4). ⊕ K0A0S9: 19.8 kDa , Q2XNL7: 20 kDa, and W8P1J2: 22.3 kDa. 10 17 26 34 43 55 72 kDa 1 2 3 4 5 6 7 19.8 kDa 20 kDa 22.3 kDa Fig. 4 Successful protein expression of transfected allergens in Western blot. Lane 1: protein ladder; Lane 2: negative control, Lane 3-7: transfected T. solium allergens . Conclusion ✓ Stably transfected chimeric pig/rat FcεRIα reporter system has been created (Fig.5) ✓ Successful cloning and sequence verification of four recombinant T. solium allergens in expression vector ✓ Successful recombinant expression Fig. 5 Stable transfection and PCR confirmation of chimeric pig/rat FcεRIα cell line. Fluorescence microscopy showing red fluorescence from transfected cells. In PCR, RBL-2H3 cells used for negative control (SS-nc). SS: chimeric pig/ rat FcεRIα, MW is a 100 bp ladder. Outlook ● Further expression and purification of candidate diagnostic allergens. ● Validation and assessment of Porcinized RBL reporter system with diagnostic allergens. ● Screening of infected and non- infected pig sera with reporter system. ● Determination of specificity and sensitivity. Commissioned by the German Federal Ministry for Economic Cooperation and Development (BMZ) and carried out by ATSAF e.V. on behalf of the Deutsche Gesellschaft für Internationale Zusammenarbeit (GIZ) GmbH.