The Production of Recombinant single chain Antibody Fragments for the Detection of Illicit Drug ...

380 Pages·2012·8.96 MB·English

The Production of Recombinant single chain
Antibody Fragments for the Detection of Illicit
Drug Residues
A Thesis submitted for the degree of
Doctorate of Philosophy
By
Joanne Brennan B. Sc. (Hons)
May 2005
Under the supervision of Professor Richard O’Kennedy
Based on Research carried out at
School of Biotechnology,
Dublin City University,
Dublin 9,
Ireland.
This thesis is dedicated to my Dad and Bernie.
Declaration
I hereby certify that this material, which I now submit for assessment on the
programme of study leading to the award of Doctor of Philosophy, is entirely my own
work, and has not been taken from the work of others, save to the extent that such
work is cited and acknowledged within the text of my work.
S igned ■
Acknowledgements
I would like to thank Prof. Richard O’Kennedy for his guidance and support over the
past few years. I would also like to thank all the members of the Applied
Biochemistry Group, both past and present, for their friendship and encouragement
throughout the duration of my Ph.D ..
I would especially like to thank all ‘the girls’ in the second floor annex for all the
great nights out. Thanks to Lynsey, Liz, Sharon and Sinead for their great friendship
over the past few years. For all the coffee breaks and for the many times when some
wine was in order and a spare bed was needed.
I would like to express my gratitude to Dr. Andrew Porter, who gave me the
opportunity to work as part of The Molecular Cell Biology Group in the University of
Aberdeen. The kindness and hospitality of this lab group was greatly appreciated.
Many good friendships that were made during this visit will not be forgotten.
Finally, I would like to thank my family for all the support they have given over the
past number of years. I would like to thank Mark for all the enouragement he gave
me throughout the course of my Ph.D., without which I never would have stuck it out.
I would especially like to thank my Dad and Bernie for everything they’ve done for
me, for their constant support and encouragement, both emotional and financial, and
especially for putting up with me during the past few months. Without them I never
would have made it this far.
Presentations
Brennan, J. (2004). The production and characterisation of scFv antibody fragments
to illicit drugs. Research collaboration. School of Molecular and Cell Biology,
University of Aberdeen, 12th February.
Brennan, J. and O’Kennedy, R. (2003). The production of novel genetically-derived
scFv antibodies capable of illicit drug detection. Defence Science and Technology
Laboratory collaboration meeting, DCU, 30th October.
Brennan, J. (2003). The production of genetically-derived scFv and bifunctional
antibody fragments capable of detecting illicit drug residues. The School of
Biotechnology Seminar Series, DCU, 10th March.
Brennan, J. and O’Kennedy, R. (2003). BIAcore technology. Antibody Training
Course, DCU, 12th February.
Brennan, J. (2002). The production of genetically-derived scFv and bifunctional
antibody fragments capable of detecting illicit drug residues. Amersham Biosciences
Workshop: From Gene to Functional Protein, Paris, 10 June.
Brennan, J. (2002). The production of genetically-derived scFv antibodies for the
detection of illicit drug residues. The National Centre for Sensor Research Cluster
seminar, DCU, 23rd May.
Abstract
Recombinant antibodies represent a more sensitive and specific detection tool for
immunoanalysis. The research carried out for this thesis describes the production of
genetically-derived single chain antibody fragments to detect illicit drugs.
A variety of novel recombinant antibody fragments against morphine-3-glucuronide, a
metabolite of heroin has been produced. A monomeric, dimeric and enzyme-labelled
scFv were characterised with respect to their binding abilities and cross reactivities.
Monomeric scFv was successfully applied to a competitive ELISA format for the
detection of morphine residues in saliva. The assay was used to positively identify
morphine residues in the saliva of drug addicts attending a rehabilitation clinic. An
inhibition assay to detect morphine in saliva was also developed using the BIAcore
3000 instrument, a surface plasmon resonance-based biosensor for detection of
biomolecular interactions in ‘real-time’. A novel sol particle lateral flow
immunoassay was generated using monomeric and dimeric scFvs for the detection of
M3G.
Recombinant antibody fragments against tetrahydrocannabinol were isolated from a
naive human library by a process known as biopanning. Phage-displayed antibodies
have been fully characterised with respect to their binding capabilities and were
successfully applied to an immunoassay for the detection of marijuana residues in
saliva.
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Abbreviations
A Absorbance
AP Alkaline phosphatase
APC Antigen presenting cell
BCA Bicinchoninic acid assay
BCIP/NBT 5-bromo-4-chloro-3-indolyphosphate/nitro blue tetrazolium
chloride
BgG Bovine-5-globulin
BIA Biomolecular interaction
BSA Bovine serum albumin
BtG Bovine thyroglobulin
C Constant
Ca Circa
CAP FUDT College of American pathologists forensic urine drug testing
cDNA Complementary deoxyribonucleic acid
CDR Comlementarity determining region
CFU Colony forming units
Ch Constant heavy chain
Cl Constant light chain
CM Carboxy-methyl
Cone Concentration
C.V. Coefficient of variation
D Diversity
DMSO Dimethylsulphoxide
dsFv Disulphide stabilised variable fragment
DTT Dithiothreitol
E Exponential
EDC N-ethyl-N-(dimethyl-aminopropyl) carbodiimide hydrochloride
EDTA Ethylenediaminetetra-acetic acid
ELISA Enzyme-linked immunosorbent assay
EMIT Enzyme multiplied immunoassay technique
EU European Union
Fab Antigen binding fragment
Fc Antibody constant region
FCA Freund’s Complete Adjuvant
FICA Freund’s Incomplete Adjuvant
Fd Antibody fragment consisting of one Vh and one Chi domain
fd A strain of filamentous bacteriophage
Ff Family of filamentous bacteriophage
Fv Antibody variable fragment
GC Gas chromatography
Gly Glycine
H Heavy chain
HAT Hypoxanthine aminopterin thymidine
Hb Heavy chain back primers
HBS Hepes buffered saline
Hf Heavy chain forward primers
HGPRT Hypoxanthine guanine phosphoribosyl transferase
His Histidine
HPLC High performance liquid chromatography
HRP Horse radish peroxidase
HUCK Human kappa constant domain
IDA Iminodiacetic acid
IFN Interferon
IgG Immunoglobulin class G
IgA Immunoglobulin class A
IgD Immunoglobulin class D
IgE Immunoglobulin class E
IgM Immunoglobulin class M
IL Interleukin
IMAC Immobilised metal affinity chromatography
IPTG Isopropyl-p-D-galactopyranoside
J Joining
Ka Equilibrium association affinity constant
Ka Association rate constant
vili
Kd Equilibrium dissociation affinity constant
Kd Dissociation rate constant
KLH Keyhole limpet haemocyanin
L Light chain
LB Luria-Bertani
Lb Light chain back primers
LDD Least detectable dose or lowest detectable dose
LE Low expression
LED Light emitting diode
Lf Light chain forward primers
LFIA Lateral flow immunoassay
Ln Natural log
LOD Limit of Detection
Log Logarithm
LOQ Limit of quantification
M Slope
Mab Monoclonal antibody
MAM Monacetylmorphine
MBDB 3.4-methylenedioxyphenyl-2-butanamine or alternatively
known as (N-methyl-l-(l,3 benzodioxol-5-yl)butanamine
MDA 3.4-methylenedioxyamphetamine
MDEA 3.4-methylenedioxy-N-ethylamphetamine
MDMA 3.4- methylenedioxymetamphetamine
MHC Major histocompatability complex
MPBS Marvel™ PBS
mRNA Messenger ribonucleic acid
MS Mass spectroscopy
M.W. Molecular weight
M3G Morphine-3-glucuronide
NE Non-Expression
NEB New England Biolabs
NHS N-hydroxysuccinimide
NTA Nitrotriacetic acid
OD Optical density
0-PD o/V/jo-Phenylenediamine
OVA Ovalbumin
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffer saline
PBST Phosphate buffered saline-tween 20
PCC Pyridinium chlorochromate
PCR Polymerase chain reaction
PDB Protein data bank
PEG Polyethylene glycol
pH Log of the hydrogen ion concentration
Isoelectric point
pi
RAG Recombinase-activating gene
RIA Radioimmunoassay
RNA Ribonucleic acid
RSS Recombination recognition sequences
RT-PCR Reverse transcription polymerase chain reaction
S Switch
SAMHSA Substance abuse and mental health service admir
scAb Single chain antibody
scback Single chain back primer
S.D. Standard deviation
S.E. Standard error
Ser Serine
scfor Single chain forward primer
scFv Single chain variable fragment
SDS Sodium dodecyl sulphate
SOC Super optimal catabolites
SOE Splice by overlap extension
StAb Stabilised single chain antibody
S/P Saliva:plasma ratio
SPR Surface plasmon resonance
TAE Tris acetic acid ethylenediaminetetra-acetic acid
TB Terrific Broth
TBS Tris buffered saline