Alkhalifah, Mohammed Ali (2007) Recombinant antibodies against Clostridium difficile toxin A. PhD

244 Pages·2010·29.04 MB·English

Alkhalifah, Mohammed Ali (2007) Recombinant antibodies against
Clostridium difficile toxin A. PhD thesis.

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Recombinant
antibodies against
Clostridium difficile Toxin A
University
Of
Glasgow
Faculty Biomedical Life Sciences
of and
Division Infection Immunity
of and
June 2007
Mohammed Ali AlKhalifah
Supervisor:
Dr. R. Aitken
A thesis for the degree
submitted
of
Doctor
of ý, j6hilosqphy
Author's declaration
This thesis is the the
original work of author except where otherwise stated
Mohammed A. AlKhalihfah
Acknowledgments
I best Dr. Robert
thanks to
express my gratitude and most sincere my supervisor
Aitken for his
continued encouragement, supervision and profound enthusiasm,
discussion, this thesis.
stimulating patience and reading of, and commenting on
I help Dr. Gill Douce
greatly appreciate and acknowledge the of as my assessor
for her helpful
advice.
I to thank Mehdi Golchin Subash Verma for helpful discussions,
wish and
friendliness humour.
and
I
thak
must sincerely my parents, my wife and my children whose support and
have helpted
thoughout
encouragement me my studies.
,7
1 thank friend Ali Abdul Jabbar Al Homood for his
also my cusin and efforts and
before during
this
continuing support and work.
My thanks to infection immunity for their help
also go all and staff members and
during
the
collaboration work
Finally, I thank the Ministry Health, Saudi Arabia for financial
of my support and
to this
allowing me carry out research work.
Table Contents
of
Chapter I
General Introduction
I General Introduction
3
1.1 Pathogenicity
8
1.2 Toxin
structure
1.3 Toxin
mode of action
9
1.3.1 Receptor binding
and receptor
11
1.3.2 Toxin A
to the
entry cell
12
1.3.3 Toxin function
A- 15
difficile Toxin
1.4 Humoral inumme Clostridium
and cellular response against
16
1.5 Methods for
control
16
1.5.1 Antibiotics
18
1.5.2 Immunotherapy
22
1.6 Toxinotypes importance
and strains of
26
1.7 Antibodies CDR
and conformation
27
1.8 Engineered
antibodies
28
1.8.1 Humanization
of antibodies
--
1.8.1.1 Chimeric
antibodies
_28
29
1.8.1.2 CDR-
grafted antibodies
29
1.8.1.3 Transgenic Antibodies
30
1.9 Recombinant Antibodies display
and phage
33
1.9.1 Creation libraries
of antibody
33
1.9.1.1 Immune libraries
-1
33
1.9.1.2 Non-immune libraries
34
1.9.1.3 Synthetic libraries
35
1.9.2 Formats for in display
antibodies phage
36
1.10 Phage display
41
1.10.1 Selection Phage
of antibody
42
1.10.2 Screening
of phage antibodies
43
1.10.3 Antibody Expression
1.10.4 Affinity
maturation
_44
45
1.11 Aims
46
Chapter
2
46
MaterWs
and methods
47
Materials
and methods
47
2.1 Bacterial
strain
47
2.2 Confirmation insert Toxin A C-terminal
of encoding region
48
2.3 Growth bacteria the Toxin A
of and expression of carboxy-terminal region
_
48
2.4 Purification the by
of protein affinity chromatography
49
2.5 Protein
assay
49
2.6 SDS-PAGE
analysis
50
2.7 Electroblotting
2.8 Recognition in ELISA 50
51
2.9 Binding
Toxin A to
of native and recombinant cellular receptors
52
2.10 Haemagglutination Toxin A
activity of recombinant
52
2.11 Sensitivity F9 Vero lines to Toxin A
of and cell native
.
IV
53
2.12 Kinetics Toxin A F9 Vero
of action of native on and cell
Toxin
A
2.13 Inhibition Toxin A and recombinant
of native with polyclonal anti-Toxin
53
A in
vitro
54
2.14 Inhibition Toxin A Fab fragments
of native with polyclonal
54
2.14.1 Digestion A
of polyclonal anti-Toxin antibody with papain
54
2.14.2 SDS-PAGE
gel electrophoresis
54
2.14.3 Electroblotting
2.14.4 Detection Toxin A by Fab fragment in ELISA
of native
_55
55
2.14.5 Inhibition Toxin A Fab fragments in
of native with polyclonal vitro
57
2.15 Tomlinson libraries
scFv
61
2.15.1 KM13 helper
phage,
63
2.15.2 Objectives
63
2.15.3 Tomlinson Library 1
63
2.15.3.1 Propagation KM13 helper
of phage
64
2.15.3.2 Titration KM 13 helper
of phage stock
65
2.15.3.3 Growing library
the
65
2.15.3.4 Titration libraries
of phage
66
2.15.3.5 Growing TG1 HB2151
and cells
66
2.15.4 Experiments before
selection
66
2.15.4.1 Growing
positive controls
66
2.15.4.2 Production
of soluble positive control
2.15.4.3 Detection by Western blot 67
of anti-BSA and anti-ubiquitin scFvs
67
2.16 Antibody
selection
2.16.1 Panning library I C- terminal domain of Toxin A 67
against purified recombinant
67
2.16.1.1 First
round of selection
68
Infecting TG I
cell with eluted phage antibodies
69
Rescue
of selected phage antibodies
69
Titration
of rescued phage
70
2.16.1.2 Second
round of selection
70
2.16.1.3 Third
round of selection
70
2.16.2 Screening by ELISA
phage
1
70
2.16.2.1 Screening by ELISA (polyclonal ELISA)
phage particles
71
2.16.2.2 Screening by ELISA (monoclonal ELISA)
phage particles
71
Monoclonal Phage ELISA
U__
2.17 Production fragments 72
of soluble antibody
2.18 Genetic 74
characterization of selected scFvs
74
2.18.1 Plasmid digestion
2.18.2 Sequence 74
of selected clones
2.19 SDS-PAGE Western blot 75
and analysis of selected clones
2.20 High level 75
expression and purification of soluble scFv
2.20.1 Purification by 76
the
of scFvs affinity chromatography
-
2.21 Biological immunochemical 77
activity and characterization of purified scFv
_
2.21.1 Recognition Toxin A by 77
of native selected scFv
2.21.2 Haernagglutination inhibition 77
assay
2.21.2.1 Preparation Rabbit 78
of erythrocytes
2.21.2.2 Haernagglutination inhibition 78
2.21.3 Rabbit haemolysis 78
erythrocyte assay
2.21.4 Indirect immunofluorescent 79
fixed
test
antibody with cells
2.21.5 Indirect immunofluorescent 80
in
test
antibody with cells suspension
_
2.21.6 SDS-polyacrylamide 81
Western blotting
gel electrophoresis and
2.21.7 Expression levels 82
of selected scFvs
2.22
Epitope 82
analysis
2.22.1
Epitope 83
saturation curve
2.22.2
Epitope 84
different
saturation experiment using two scFvs
2.22.3
Blocking 86
by
of monoclonal phage antibody epitopes scFv of the same specificity
_
2.22.3.1 Preparation 86
of phage antibodies
2.22.3.2 Optimization 87
blocking by ELISA
of epitope
2.22.3.3 88
Blocking
by
of phage antibody
scFv
V
88
2.23 Assay for toxin
neutralizing activity
88
2.23.1 Flow binding
toxin
cytometric analysis of
89
2.23.2 Inhibition Toxin A in
of cytotoxicity vitro
89
2.23.2.1 Preparation F9 line
of cell
89
2.23.2.2 Addition Toxin A-
of antibody mixtures
90
2.24 Epitope
mapping
90
2.24.1 Materials
and methods
2.24.1.1 Bacteria
and plasmids
90
2.24.1.2 Preparation
of pET767
91
2.24.2 Polymerase
chain reaction
,
91
2.24.2.1 PCR
primers
94
2.24.2.2 PCR
reactions
95
2.24.3 Elution DNA from
and purification of agarose gels
95
2.24.4 Restriction digestion ligation
enzyme and
96
2.24.5 DNA
sequencing
98
2.24.6 Cloning
98
2.24.6.1 Cloning into 2.1
pCR vector
-TOPO
98
2.24.6.2 Cloning into
pCG806
98
2.24.6.3 Ligation
99
2.24.6.4 Transformation
99
2.24.6.5 Preparation DNA duplex
of synthetic
99
2.24.6.6 Phosphorylation
of annealed oligonucleotides
100
2.25 Protein
expression
100
2.25.1 SDS-PAGE
and electrobloting
102
Chapter 3
102
Results
103
3 Results
3.1 Confirmation Toxin A C-terminal binding domain 103
of sequences encoding
-
3.2 Purification the by 105
of protein affinity chromatography
3.3 SDS-electrophoresis 105
ý
3.4 Electroblotting 107
analysis
3.5 Recognition in ELISA 107
3.6 Binding Toxin A to 110
of native and recombinant cellular receptors
3.7 Haemagglutination Toxin A 110
activity of recombinant
3.8 Sensitivity F9 Vero lines Toxin A 113
to
of and cell native
3.9 Kinetics Toxin A F9 Vero lines 113
of action of native on and cell
3.10 Inhibition 114
the Toxin A
of action of native
3.11 Inhibition Toxin A Fab fragments 119
of native with polyclonal
3.11.1 Digestion 119
A
of polyclonal anti-Toxin antibody with papain
3.11.1.1 SDS-PAGE
gel electrophoresis
3.11.2 Electroblotting 122
3.11.3
Detection Toxin A by Fab fragment in ELISA 122
of native
3.11.4
Inhibition Toxin A Fab fragments in 125
of native with polyclonal vitro
3.12 Tomlinson library 125
scFv
3.12.1
Growing library 125
the
3.12.2
Detection 127
by Western blot
of anti-BSA and anti-ubiquitin scFvs
3.12.3
Phage display against the C-terminal domain Toxin A 127
of
3.12.4
Screening 130
by ELISA
polyclonal phage
3.12.5
Screening 130
by ELISA
monoclonal phage
3.12.6
Screening 133
by ELISA
monoclonal soluble scFv antibodies
3.13
Genetic 135
characterization of selected scFvs
.
3.14
Sequence 135
of selected clones
136
3.15 Detection Toxin A by
of native selected scFv
143
3.16 Haernagglutination inhibition
143
3.17 Haemolysis
assay
145
3.18 Indirect immunofluorescent fixed
test
antibody with cells
3.19 Indirect immunofluorescent 147
test
antibody with suspended cells
3.20 Characterization icFvs 149
of selected
3.20.1 SDS-polyacrylamide Westem blotting 149
gel electrophoresis and
3.21 Expression levels 149
of selected scFvs
3.22 Purification the by 152
of scFvs affinity chromatography
3.23 Epitope 153
saturation curve
3.24 Epitope two different 153
saturation experiment using scFvs
3.25 Competitive ELISA 157
with monoclonal phage antibodies and scFv
3.25.1 Preliminary 157
experiments
3.25.2 Competitive ELISA 160
experiments
3.26 Flow Toxin A-receptor interaction 162
cytometric analysis of
3.27 Inhibition Toxin A by 164
of cytotoxicity scFvs
3.28 Epitope 167
mapping
3.28.1 Cloning three Toxin A into 2.1 167
of regions of pCR vector
-TOPO
3.28.1.1 Restriction 167
analysis
3.28.2 Cloning Toxin A fragments into 171
a maltose-binding protein vector
3.28.3 Cloning DNA linker into MBP 171
of pCG806 vector
3.29 Characterisation MBP fusions 177
of
Chapter 4 1025
4 Discussion 185
.
4.1 Purification C-terminal domain Toxin A
and characterization of recombinant of
185
4.2 Screening the Tomlinson library 192
of
4.3
Biological A 195
activity of anti-Toxin scFvs.
4.4 Epitope
A 200
mapping of anti-Toxin scFv
4.5 Conclusion
202
5 Appendices
204
5.1
Luria-Bertani (LB) broth 204
or agar
5.2 Preparation
204
of antibiotic solutions
5.3 SOC
204
medium
5.4 Citric
buffer 205
acid phosphate
6
Bibliography
206
List tables
of
24
Table 1.1: RFLP for
typing toxin
characteristics used of genes
Table 1.2: C. difficile 25
toxin
strains with variant genes
1-
Table 2.1: Inhibition Toxin A Fab fragments in
of native with polyclonal vitro
design: 56
experimental
Table 2.2: Design 85
of epitope saturation experiment
Table 2.3: Bacterial for 92
strains used epitope mapping
Table 2.4: List for
of primers used epitope mapping and characterization of scFv
93
genes
Table 2.5: List 97
their target
of restriction enzymes and sequences
Table 3.1: Recoveries during 129
of phage selection
Table 3.2 List the the in
: of primer pairs and predicted size of products used cloning
four Toxin A 168
of regions of
Table 3.3: List
the detected fragments 183
of scFvs and
List figures
of
14
Figure 1.1: Summary Toxin A
the
of proposed mode of action of
32
Figure 1.2: The formats
various of engineered antibodies
Figure 2.1: Structure 59
of phagemid pIT2
60
Figure 2.2: DNA
sequence of pIT2 vector
Figure 2.3 Principle (Goletz 2002) 62
: of proteolytic selection et al.,
Figure 3.2: Complete Toxin A from
nucleic acid sequence encoding recombinant
104
pET767
Ll-!
-
Figure 3.3: SDS-PAGE Toxin A by
analysis of recombinant affinity chromatography
106
Figure 3.4: Western blotting Toxin A by
analysis of recombinant purified affinity
108
chromatography
Figure 3.5: Detection Toxin A in ELISA. 109
of recombinant
Figure 3.6: Immunofluorescence detect binding
to the
assay of recombinant and
Toxin A F9 ill
to
mative receptors on cells
Figure 3.7: Haemagglutination Toxin A 112
activity of recombinant
Figure 3.8: Sensitivity F9 lines to Toxin A 115
of cell native
Figure
3.9: Sensitivity
F9 lines to Toxin A 116
of cell native
Figure
3.10: Sensitivity Vero lines to Toxin A 117
of cell native
Figure
3.11: Effective
time the Toxin A F9
of exposure on action of native on and
'Yero 118
cells.
Figure
3.12:
Inhibition Toxin A by A 120
of polyclonal anti-Toxin and rTA
Figure
3.13: Non
SDS-PAGE
digested A
reducing
analysis of papain anti-Toxin
antiserum. 121
Ll-
Figure
3.14: Western
blotting
digested Toxin A
analysis of papain anti antiserum
-123
Figure
3.15: Reaction
intact
IgG Fab fragments ELISA
of Toxin A in
and
with native
r 124
Figure
3.16:
Inhibition
Toxin A by
digested A
of
papain
polyclonal anti-Toxin
antiserum.
126
,
Figure
3.17:
Polyclonal
ELISA
phage Tomlinson library I during
of against
panning
recomninantToxin A 131
,