studies on the production, characterisation and applications of monoclonal antibodies and ...

412 Pages·2013·8.86 MB·English

STUDIES ON THE PRODUCTION,
CHARACTERISATION AND
APPLICATIONS OF
MONOCLONAL ANTIBODIES AND
ANTIBODY-BASED
CONJUGATES.
A dissertation submitted for the degree of Ph.D.
by
Dolores J. Cahill.
Under the supervision of
Professor Richard O’Kennedy,
December, 1994.
School of Biological Sciences,
Dublin City University, Dublin 9, Ireland.
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ABSTRACT
The work described in this thesis involved the production of antibodies to several defined antigens and
the chemical synthesis of bispecific antibodies
Monoclonal antibodies against glial fibrillary acidic protein (anti-GFAP), and against a GFAP-like protein
on the human anaplastic astrocytoma cell line, G-CCM (anti-G-CCM) were produced by electrofusion and
by fusion using polyethylene glycol, respectively These antibodies were purified with saturated ammonium
sulphate (S AS) and protein A These antibodies were fully characterised by ELISA, Isotyping, SDS-PAGE,
Immunocylochemisiry, Dot Blot, Western immunoblotting, BIAcore, HPLC, Immunofluoresence and
FACS An attempt was made to clone and express the variable regions of the anti-G-CCM monoclonal
antibody in Escherichia coh
Iodinated Bolton-Hunter reagent (IBHR) was conjugated to bovine serum albumin (BSA), ovalbumin and
keyhole limpet haemocyanin (KLH) These IBHR-protein conjugates were characterised by the iodide
assay and the Bradford protein assay The IBHR-BSA conjugate was used to immunise six mice and a
rabbit to produce anti-IBHR antibodies A monoclonal anti-IBHR antibody was not obtained However,
a polyclonal anti-IBHR antibody was produced and was purified by SAS, protein A and CNBr-activated
sepharose affinity chromatography This antibody was characterised by ELISA, SDS-PAGE and HPLC
Bispecific antibodies were chemically generated using (1) anti-GFAP and ana-IBHR and (u) anti-G-CCM
and anti-IBHR Ricin is a highly cytotoxic compound Bispecific antibodies were made using an acquired
anti-Ricm A chain monoclonal antibody together with anti-G-CCM and anti-GFAP These bispecific
antibodies were characterised by ELISA, SDS-PAGE, HPLC and FACS and may be used to target
radioactive compounds and toxins to astrocytoma cells, in vivo
A catalytic monoclonal antibody was generated using a transition state analogue-KLH conjugate as an
immunogen in six mice This antibody was precipitated using sodium sulphate and purified by protein G
affinity chromatography This antibody catalysed the hydrolysis of the corresponding carbonate and its rate
kinetics were determined
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1
DECLARATION
I hereby certify that this material, which I now submit for assessment on the programme of study leading
to the award of Ph.D. is entirely my own work and has not been taken from the work of others, save and
to the extent that such work has been cited and acknowledged within the text of my work.
DEDICATION
To my mother
,
V
and the memory of my father
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111
ACKNOWLEDGEMENTS
I would like to thank my supervisor, Prof Richard O’Kennedy, for his support throughout my Ph D
research Special thanks are due to Dr Ken Carroll for his helpful advice and interest during this project
and to whom I am grateful
I am especially indebted to Ms Carolyn Wilson for her expert technical assistance in caring for the
animals used in this project and for her support through the many difficulties encountered Dr Liz Moran
is appreciated for her assistance with Western immunoblotting and for her interest and advice with the
problems experienced during the production of monoclonal antibodies
I thank Prof Martin Clynes for the use of the facilities at the National Cell and Tissue Culture Centre
I would like to thank all the members of the Applied Biochemistry Group, past and present, for their
friendship, kindness and assistance
I am indebted to Dr Mark Searcy, U C D , for his useful suggestions and discussions regarding the
catalytic antibody work I am grateful to Dr Gerry Gallacher, Umversity of Brighton, for supplying me
with the chemical compounds and polyclonal antibody used in the catalytic antibody work
I acknowledge Dr Ellen Vitetta, University of Texas Southwestern Medical Centre, for allowing me to
visit her laboratory and facilitating my research with ncin Permission to use the anti-ncm A chain
antibody was gratefully obtained from Dr Patrick Trown, Xoma Corporation, California and Dr Malcolm
V Pimm, University of Nottingham, supplied this antibody The technical expertise of Mrs Kate Cotter,
University of Maynooth, during the FACS analysis is appreciated
I would like to thank the members of the John Barry Building, especially Fiona, Geraldine and Margaret,
for their assistance with the molecular biology aspects of this research
Special thanks are due to the ’Good Time Club’ for ensuring life was never dull D C U rowing club is
especially appreciated for lots of happy times at Islandbndge and for many enjoyable weekends away
Finally, I would like to thank John for his unfailing support, encouragement and assistance throughout this
Ph D , especially during the writing of this thesis
IV
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PUBLICATIONS
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Prosser, E , Cahill, D and O’Kennedy, R , (1991)
’Antibody-mediated in vivo detection and treatment of disease ’
J Biomed Sciences, 1 145-152
O’Kennedy, R , Bator, J , Reading, C , Nolan, O , Quinlan, N , Cahill, D and Thomas, M W (1993)
’Recent novel applications of antibodies in analysis Production and applications of lodinated conjugates
for use in antibody screening, isolation and detection procedures ’
Reviews on Anal Chem, pps 335-348
Cahill, D , Roben, R , Quinlan, N and O’Kennedy,¡R
’Production of antibodies ’ '
Invited chapter for The Encyclopedia of Analysis, in press
J
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Cahill, D , Moran, L M and O’Kennedy, R
’Production and characterisation of a monoclonal antibody against the human astrocytoma cell line G-
CCM, which also binds GFAP, an astrocytoma associated antigen ’
In preparation
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ABBREVIATIONS
A Absorbance
ADCC Antibody-dependent cell-mediated
ATP Adenosine tnphosphate
B-Cell B lymphocyte cell
BCA Bicinchonimc acid
BCIP 5-bromo-4-chloro-3-indoy 1-phosphate
BHR Bolton-Hunter reagent
(3-(4-hydroxyphenyl)propiomc acid N-hydroxysuccmimide ester)
BSA Bovme serum albumen i
C Constant
CD Cluster of differentiation
CDC Complement-dependent cytotoxicity
CEA Carcinoembryomc antigen
CDC Complement dependent cytolysis
cDNA Complementary DNA
CDR Complementarity-determining region
CFA Complete Freund’s adjuvant
conc Concentration
CTP Cytidine tnphosphate
cv Column volume
D Diversity
DEPC Diethylpyrocarbonate
dH20 Distilled water
ddH20 Double distilled water
DMEM Dulbecco’s modification of Eagle’s medium
DMEMS10 DMEM supplemented with 10% (v/v) FCS
DMF N,N'-dimethylformarmde »
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DMSO Dimethylsulphoxide
DNA Deoxyribonucleic acid ,
DNP 2,4,-di-nitro-phenol
dNTP Deoxynbonucleoside triphosphate
DPX Distrene, dibuthyl phthalate, xylene
DTT Dithiothreitol !
EDTA Ethylene diamine tetra-acetic acid
ELISA Enzyme-linked immunosorbent assay
EtBr Ethidium bromide
F(ab) F(ab) fragment of immunoglobulin
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F(ab02 F(ab02 fragment of immunoglobulin
FACS Fluorescence activated cell sorting
Fc Antibody constant region
FCS Foetal calf serum
FITC Fluorescein-isothiocyanate
GFAP Glial fibrillary acidic protein
GTP Guanosine triphosphate
H Heavy chain
HA Hypoxanthine 8-azaguanine
HAMA Human anti-mouse antibody
HAT Hypoxanthine aminoptenn thymidine
H/E Haematoxyhn and Eosin stain
HEPES N- [2-hydroxyethyl]piperazine-N/-[2-ethanesulphomc acid]
HIPO HEPES, insulin, sodium pyruvate, oxaloacetic acid
HPLC High pressure liquid chromatography
HRP Horse radish peroxidase
HT Hypoxanthine thymidine
131I Iodine-131
IBHR Iodinated Bolton-Hunter reagent
IFA Incomplete Freund’s adjuvant
Ig Immunoglobulin
IL-6 Interleukin-6
IMEM Iscove’s DMEM
inIn Indium-111
iP Intrapentoneal
1 V Intravenous 1
J Joining
K Kappa light chain 1
KLH Keyhole limpet haemocyamn
L Light chain
LB Luna Bertam broth
LMP Low melting point
mab Monoclonal antibody
MHV Mouse hepatitis virus
mRNA Messenger RNA
MWM Molecular weight markers
n Number of replicates j
NA Not applicable
NBT Nitro blue tetrazolium
Vll
ND Not determined
NRK Normal rat kidney
OD Optical density
ONPG O-nitrophenyl galactopyranoside
OPD O-phenylenediamine
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffered saline (Dulbecco’s A)
PCR Polymerase chain reaction
PEG Polyethylene glycol
PMSF Phenyl-methyl-sulphonyl fluoride
PVC Polyvinyl chloride
RES Reticuloendothelial system
RNA Ribonucleic acid
RNase Ribonuclease
rpm Revolutions per minute ,
RT Reverse transcriptase
RXN Reaction
SAR Structure-activity-relationship
SAS Saturated ammonium sulphate
SD Standard deviation
SDS Sodium-dodecyl sulphate
SPDP N-succinimidyl-3-(2-pyndyldithiol)propionate
SPR Surface plasmon resonance
Taa Tumour-associated antigens
TAE Tns-acetate-EDT A
Tag Thermus aquaticus
TBE T ns-borate-EDT A
TBS Tns buffered saline
"Tc Technetium-99
T cell T lymphocyte
TCM T-cell-conditioned medium
TE TRIS-EDTA
TEMED N^N'JM'-tetramethyl-ethylaminediamine
TRIS Tns(hydroxymethyl)methylamine
TIP Thymidine triphosphate
upH20 Ultrapure water
UV Ultraviolet
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V Variable
wt weight
vm
UNITS
g gravity
hr(s) hour(s)
kD kiloDalton
mg milligram
min minute(s)
ml millilitre(s)
M Molar
mM millimolar
Pg microgram
pM micromolar
N normal
ng nanogram
nm nanometre
v/v volume per volume
w/v weight per volume