Prsentation PowerPoint

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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
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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
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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.

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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.

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To address these problems Recombinant DNA
Technology has provided new facilities
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.
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• 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.
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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.
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Cloning of the scFv50AD1 gene
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Cloning scFv50AD1-AP fusion protein into pLIP6
vector
Sequencing and screen against databases
allowing the periplasmic exportation of the
fusion protein

• This facilitates
• disulfide bond formation
• solubility
• extraction
• and purification of proteins

The nucleotide and deduced amino acid sequences
of the scFv50AD1
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Expression and purification analyses

• TG1 bacterial transformation
• induction
• periplasmic proteins extraction
• and purification

Lane 1 crude preparation periplasmic Lane 2
purified fusion protein Lane 3 non-induced cell
culture
1 2
1 2
M 1 2 3
97
97
scFv-AP
66
66
45
45
30
30
20.1
20.1
Western blot
Directly revealed with BCIP/NBT AP substrate
SDS PAGE 12 - silver-staining
Treated with Anti-AP antibody Anti mouse-IgG-HRP
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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
• and the antigen-binding activity
• against the RVG

The corresponding colorimetric signal increased
in a dose-dependent manner with increasing
amounts of scFv50AD1-AP
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• 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

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• 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)

• The results showed that
• 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.

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• 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

• 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
• 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.

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• 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
The interaction was analysed by colorimetric
BCIP/NBT AP substrate

• Mouse brain impressions with RV infection
• blocked with 20 mM levamisol,
• The scFv50AD1-AP fusion protein were added and
  incubated

• 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
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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.

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Acknowledgements
Imène Turki Pr. Koussay Dellagi   Groupe
Immuno-Biotechnologie LIVGM, Institut Pasteur
de Tunis
Mohamed Saadi Dr. Habib Kharmachi 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