Download Genetically engineered single-chain antibody fusion proteins

Genetically Engineered Single-Chain Antibody
Fusion Proteins
for Detection of Rabies Virus Antigen
Dr Mohamed MOUSLI
Groupe Immuno-Biotechnologie
Laboratoire LIVGM
Institut Pasteur de Tunis
The most widely used tests for the
detection of rabies antigens are
 Flurorescent antibody test (FAT)
 Immunohistochemistry
 Enzyme-linked immunosorbent
assay (ELISA)
These tests are
easy,
sensitive,
currently recommended by the WHO
Expert Committee on Rabies
However, these routine laboratory tests
present drawbacks:
 requires expensive reagents and instruments;
 the procedures are relatively long;
 well-trained technicians;
 is carried out with primary or secondary antibodies,
 that are labeled with sensitive
reporter molecules,
 like fluorescent dyes
 colorimetric enzymes
 the chemical labelling is the conventional
method for obtaining the conjugates.
The chemical cross-linking methodology
present some difficulties
 such as a random cross-linking chemical reaction;
 is usually not specific;
 produce heterogeneous conjugates
e.g. enzyme-enzyme conjugates, antibody-antibody conjugates
 leads to side reactions that damage the combining site;
 and reduce activity;
 require several purification steps;
 sometimes producing important variations from batch to batch.
To address these problems
Recombinant DNA Technology has provided new facilities
single-chain antibody gene
proteic tracer gene
Fusion gene
Transformed
bacteria
Fusion protein
Genetic engineering has provided a way to create a chimeric bifunctional
molecules in which the variable domains of an antibody are genetically
linked to unrelated proteic tracers and produced by recombinant bacteria.
The gene fusion approach is
 simple, easy and reproducible;
 it gives a control molar ratio between antibody
and labelling group;
The recombinant immunoconjugate molecule
expressed in bacteria systems is
 rapidly grow up them on an industrial scale;
 rapidly purified in one-step;
 and with a well-controlled quality;
The genetic approach makes possible
the improvement of the antibody affinity by genetic engineering
in order to reach or exceed the sensitivity level.
Here we describe
the generation of a recombinant scFv from the 50AD1
anti-Rabies Virus Glycoprotein hybridoma,
The mouse hybridoma cell line 50AD1
 secreting a neutralizing MAb directed against the
Rabies Virus Glycoprotein
 MAb 50AD1 binds to conformational antigenic site III
its genetic fusion with an engineered bacterial alkaline
phosphatase;
and the use of this recombinant colorimetric fusion
protein (scFv-AP) in different assays for a one-step
detection of the native form of rabies glycoprotein.
Cloning of the scFv50AD1 gene
VH VL
PM scFv
Sequencing and screen
against databases
Cloning scFv50AD1-AP
fusion protein
into pLIP6 vector
allowing the periplasmic exportation
of the fusion protein
The nucleotide and deduced amino
acid sequences of the scFv50AD1
This facilitates:
 disulfide bond formation
 solubility
 extraction
 and purification of proteins
Expression and purification analyses
TG1 bacterial transformation
Lane 1: crude preparation periplasmic
 induction
 periplasmic proteins extraction
 and purification
M
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Lane 2: purified fusion protein
Lane 3: non-induced cell culture
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2
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2
scFv-AP
66
45
30
20.1
SDS PAGE 12 %- silver-staining
Western blot
Directly revealed with
Treated with Anti-AP antibody
BCIP/NBT AP substrate
Anti mouse-IgG-HRP
Bifunctionality of the recombinant fusion protein
 ELISA test
for evaluating the activity of scFv50AD1-AP fusion protein to RVG
Microtiter plates were coated with
 inactivated purified rabies virus (○)
 rabies viral glycoprotein (●),
(Platelia Rabies kit)
The bound conjugate was directly
revealed by the AP activity of the
recombinant immunoconjugate
This first result strongly indicates that
the recombinant fusion conjugate is
fully bifunctional had both
 the AP enzymatic activity
The corresponding colorimetric signal
increased in a dose-dependent manner
with increasing amounts of scFv50AD1-AP
 and the antigen-binding activity
against the RVG
 Immuno-capture ELISA test
Microtiter plates were coated
 with standard reference serum anti-rabies
 treated with various amounts concentrations of rabies
virus PV strain (▲) preparartion in cell culture
 and then with scFv50AD1-AP fusion protein
Background with the blocking solution (■)
revealed directly in one-step by
detecting AP enzymatic activity
In the presence of increasing concentrations of RV, the enzymatic
activity increased in a dose dependent manner
We showed that
 the recombinant immunoconjugate is bifunctional
 and the estimation of the quantity minimal detectable of the RVG
content of viral suspensions is about 160 ng
 Dot blot assay
Sensitivity of the chimeric scFv-AP fusion protein for the detection of RV antigen
was tested by Dot blot assay
 Two-fold serial dilutions of purified rabies virus preparation in cell culture were dotted
onto nitrocellulose membrane (from 5000 to 5 ng)
A
B
The results showed that
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NC
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NC
scFv50AD1-AP
in one-step
50AD1 Mab
in two-step
 the lower limit of RV antigen was detectable
at the concentration from 156 ng approximately;
 were comparable when we used parental
MAb in two-step procedure;
 no staining was observed in the
negative control;
 The total one-step reaction procedure take
no more than 2 h for evaluating the RV antigen
content of viral suspensions.
 Cell culture test
We developed immunocytochemistry system to detect viral antigen that can be
used with conventional light microscopy for localizing the RV in cells
 Monolayers of BHK-21 cell were infected with RV suspension;
 18 h p.i., the cells were fixed and endogenous alkaline phosphatase
was blocked with 5 mM levamisol,
 The scFv50AD1-AP fusion protein were added and incubated
The interaction was analysed
by colorimetric BCIP/NBT
AP substrate
scFv50AD1-AP
This photomicrographs showed that:
 the dark staining in cellular membrane
corresponding to immunoreactive for RV particles
 The same pattern of staining was observed
when parental 50AD1 MAb was in two-step
MAb50AD1
 no chromogenic substrates were observed NC
 This scFv50AD1-AP fusion protein
 has dual activity
 can be used for rapid and specific detection of
the rabies virus in cell culture in a one-step
procedure.
 Detection of rabies antigen in brain impressions
Like the d-FAT, Recombinant Colorimetric Immunohistochemical test was performed
on brain touch impressions to detect rabies virus antigen
but the product of the reaction can be observed by light microscopy
 Mouse brain impressions with RV infection
 blocked with 20 mM levamisol,
 The scFv50AD1-AP fusion protein were added and incubated
The interaction was analysed
by colorimetric BCIP/NBT
AP substrate
The results showed that
 the dark staining corresponding to immunoreactive
for RV particles
 no chromogenic substrates were observed NC
(uninfected brain)
 a sensitivity and specificity equivalent to those of
the d-FAT
These qualities make it ideal for testing under
Field conditions and in developing countries
In conclusion
The present work demonstrates
 that recombinant anti-RVG scFv50AD1-AP conjugate is a promising
alternative new reagent for rabies virus immunodetection in one-step
procedure;
 can be produced in homogeneous bifunctional reagent,
 easily,
 quickly,
 reproducibly
 and at low cost;
 could be used for quality control in the manufacturing process of rabies
vaccines (ELISA, IC-ELISA or Dot-blot);
 and may be used directly on a smear to confirm the presence of rabies
antigen in cell culture or in brain tissue of mice that have been inoculated
for diagnosis.
Acknowledgements
Imène Turki
Pr. Koussay Dellagi
Mohamed Saadi
Dr. Habib Kharmachi
Groupe Immuno-Biotechnologie
LIVGM,
Institut Pasteur de Tunis
Unité Spécialisée de la Rage,
Laboratoire de Microbiologie
Vétérinaire ;
Institut Pasteur de Tunis
Collaborations
Drs Christine Tuffereau and Yves Gaudin,
Laboratoire de Virologie Moléculaire et Structurale
UMR 2472 CNRS-INRA, Gif-sur-Yvette, France
Dr Frédéric Ducancel,
Département d'Ingénierie et d'Etudes des Protéines CEA-Saclay, France
Dr Philippe Billiald,
Muséum National d'Histoire Naturelle, Paris, France
This work was supported by grant from the EMRO-COMSTECH for Research
in Applied Biotechnology & Genomics in Health