Genetically engineered single-chain antibody fusion proteins for - OIE

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12 Δεκ 2012 (πριν από 4 χρόνια και 11 μήνες)

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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



Flurorescent antibody test (FAT)




Immunohistochemistry




Enzyme
-
linked immunosorbent

assay (ELISA)

The most widely used tests for the
detection of rabies antigens are




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
Fusion
protein
Transformed
bacteria
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,


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.


The mouse hybridoma cell line 50AD1




secreting a neutralizing MAb directed against the

Rabies Virus Glycoprotein




MAb 50AD1 binds to conformational antigenic site III


VH VL PM
scFv
Cloning of the scFv50AD1 gene


The nucleotide and deduced amino

acid sequences of the scFv50AD1

Sequencing and screen

against databases


Cloning scFv50AD1
-
AP
fusion protein


into pLIP6 vector

allowing the periplasmic exportation

of the fusion protein


This facilitates:



disulfide bond formation



solubility



extraction



and purification of proteins


scFv
-
AP



M
1

2 3

97

66

45

30

20.1





97

66

45

30

20.1






1 2 1 2

Expression and purification analyses



TG1 bacterial transformation





induction



periplasmic proteins extraction



and purification


Western blot

Treated with Anti
-
AP antibody
Anti mouse
-
IgG
-
HRP

Directly revealed with
BCIP/NBT AP substrate


SDS PAGE 12 %
-

silver
-
staining


Lane 1: crude preparation periplasmic

Lane 2: purified fusion protein

Lane 3: non
-
induced cell culture


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


The corresponding colorimetric signal

increased in a dose
-
dependent manner

with increasing amounts of scFv50AD1
-
AP


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



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

scFv
50
AD
1
-
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
1
2
3
4
5
6
1
2
3
4
5
6
7
8
9
10
11
NC
7
8
9
10
11
NC
A
B
1
2
3
4
5
6
1
2
3
4
5
6
7
8
9
10
11
NC
7
8
9
10
11
NC
scFv50AD1
-
AP
in one
-
step
50AD1 Mab
in
two
-
step
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.

scFv50AD1
-
AP
MAb50AD1
scFv50AD1
-
AP
MAb50AD1


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.



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



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