the development and applications of polyclonal and monoclonal antibodies for the detection of ...

327 Pages·2012·6.88 MB·English

THE DEVELOPMENT AND APPLICATIONS OF
POLYCLONAL AND MONOCLONAL ANTIBODIES FOR
THE DETECTION OF ILLICIT DRUGS IN SALIVA
SAMPLES
A thesis submitted for the degree of
Doctorate of Philosophy
by
Lorna M. Fanning B.A.(Mod), M.Sc.
September 2002
Under the supervision of Professor Richard O’Kennedy
Based on research carried out at
School of Biotechnology,
Dublin City University,
Dublin 9,
Ireland.
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 and to the extent that such work has been
cited and acknowledged within the text of my work.
Signed: j? ID No.:
Date: d \ S a p t '00.
Acknowledgements
Sincere thanks to Prof. Richard O'Kennedy for his guidance and support throughout this
project. Thanks to the following for their support and assistance:
• Members of the SMT Project Group from Envitec, Nunc, and University of Gent
• Dr. Bridin Brady, State Laboratory, Dublin
• Dr. Eamon Keenan, staff and clients of Trinity Court Drug Treatment Centre
Thanks to my lab colleagues at DCU for their help (especially Joanne for her help with
sample collection and Stephen for his help with the cells).
Thanks to my friends, especially 'the girls' for all the fun throughout the years. Thanks
also to my family Ailbhe, Joe, Carmel, Brig, Andy, Mary and Andy. Special thanks to
Mam and Dad for everything!
Publications
Fitzpatrick, J., Fanning. L., Hearty, S., Leonard, P., Manning, B.M., Quinn, J.G., and
O’Kennedy, R. (2000). Applications and recent developments in the use of antibodies
for analysis. Anal. Letts, 33:2563-2609.
Fanning, L. and O’Kennedy, R. (2002). Developments in rapid immunological-based
methods of detection of illicit drugs in saliva samples. In Preparation. TRAC
Fanning, L. and O’Kennedy, R. (2002). Development and characterisation of anti­
amphetamine and anti-methamphetamine monoclonal antibodies and application for
detection of amphetamines in saliva samples. In Preparation. J. Immunol. Meth.,
Abstract
Anti-tetrahydrocannabinol (THC), anti-cocaine and anti-morphine polyclonal antibodies
were produced. These antibodies were successfully applied to an ELISA format for the
detection of THC, cocaine, and morphine in saliva samples.
Monoclonal antibodies against amphetamine and its derivatives were produced using
two different conjugates, amphetamine-bovine serum albumin and methamphetamine-
bovine serum albumin. Two successful clones were produced, and the antibodies were
applied to an ELISA format for the detection of amphetamine, methamphetamine, and
the other common amphetamine derivatives, such as methylenedioxyamphetamine
(MDA) and methylenedioxymethamphetamine (MDMA). The ELISA was developed
using saliva as the matrix. During the screening stage of the production of these
antibodies, particular attention was given to their cross reactivity profiles. Among the
molecules tested for cross-reactivity, were legally available medications such as
ephedrine, as other commercially available antibodies show cross reactivity. The
resulting monoclonal antibodies detected amphetamine and other designer derivatives,
and showed negligible cross reactivity with the legal structurally related molecules.
The antibodies were applied to a biosensor (BIAcore) assay for the detection of
amphetamine and methamphetamine in saliva samples. The affinity constants for the
antibodies were determined by ELISA and BIAcore methods. The values obtained
were found to be similar by both methods.
A novel automated prototype device, developed by our collaborators, Envitec, was
optimised and the anti-THC polyclonal antibody was applied to it for the screening of
saliva samples for the presence of THC. This was a rapid, qualitative test, and it could
be performed in less than 20 minutes. The basis of the assay was competition between
horseradish peroxidase-labeled THC and THC present in the saliva samples, for binding
to the anti-THC polyclonal antibodies that coated the reaction wells of the device.
Abbreviations
APC antigen presenting cell
BDB benzodioxole-5-butanamine
BEC benzoylecgonine
BIA biomolecular interaction analysis
BSA bovine serum albumin
BtG bovine thyroglobulin
CDR complementarity determining regions of antibody
CE capillary electrophoresis
CV coefficient of variation
DNA deoxyribonucleic acid
EDC N-ethyl-N'-(dimethylaminopropyl) carbodiimide
EDTA ethylenediaminetetra acetic acid
EME ecgonine methyl ester
ELISA enzyme-linked immunosorbent assay
EMIT enzyme-multiplied immunoassay technique
Fab binding region of antibody above the hinge region
Fc constant region of antibody molecule
FCS foetal calf serum
FPIA fluorescence polarisation immunoassay
Fv variable binding fragment of antibody
GC/MS gas chromatography/mass spectroscopy
HAT hypoxanthine aminopterin thymidine
HPLC high performance liquid chromatography
HT hypoxanthine thymidine
HBS Hepes buffered saline
IgG immunoglobulin class G
IgA immunoglobulin class A
IgD immunoglobulin class D
IgE immunoglobulin class E
IgM immunoglobulin class M
ka equilibrium association affinity constant
ka association rate constant
vi
Kd equilibrium dissociation affinity constant
kd dissociation rate constant
MAb monoclonal antibody
MDA 3,4-methylenedioxyamphetamine
MDMA 3,4-methylenedioxymethamphetamine
MBDB 3,4-methylenedioxyphenyl-2-butanamine
MDEA 3,4-methylenedioxy-N-ethylamphetamine
MW molecular weight
NEAA non-essential amino acids
NIDA National Institute of Drugs of Abuse
NHS N-hydroxysuccinimide
OVA ovalbumin
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffer saline
PEG polyethylene glycol
pH log of the hydrogen ion concentration
SAMHSA Substance Abuse and Mental Health Service
Administration
scFv single chain Fv antibody derivative
SE standard error
SDS sodium dodecyl sulphate
SMT Standard, Measurements and Testing Framework
S/P saliva/plasma ratio
SPR surface plasmon resonance
THC tetrahydrocannabinol
THC-COOH tetrahydrocannabinol carboxylic acid
THY thyroglobulin
UV ultraviolet
Vh variable region of heavy chain
Vl variable region of light chain
WHO World Health Organisation
vii
Units
°c degrees Celcius
cm centimetres
grams
g
KDa kilodaltons
Kg kilogram
1 litre
microgram
Mg
III microlitre
pM micromolar
M molar
mg miligram
min minute
ml millilitre
mm millimetres
nm nanometre
nM nanomolar
mol molar
rpm revolutions per minute
RU response units
sec, s seconds
v/v volume per unit volume
w/v weight per unit volume
Table of Contents:
Declaration «
Acknowledgements Hi
Publications >v
Abstract v
Abbreviations vi
Units viii
Chapter 1
Introduction 1
1.1 Drugs of abuse in saliva - Background 2
1.1.1 Saliva as a matrix 2
1.1.2 pH of saliva and influence on drug concentrations 5
1.2 Other alternative biological matrices 6
1.3 Legal status of alternative biological matrices 10
1.4 Drugs of abuse: metabolism, form and concentrations 11
found in saliva
1.4.1 Cocaine 11
1.4.2. Tetrahydrocannabinol 15
1.4.3 Amphetamines 17
1.4.4. Opioids 22
1.5 Levels of detection of assays and cut-off levels 25
1.6. Methods of detection of drugs of abuse 26
1.6.1 Immunoassays 26
1.6.1.1 Competitive Immunoassay 27
1.6.1.2 Non-Competitive Immunoassay 27
1.6.2. Enzyme-Multiplied Immunoassay Technique 32
1.6.3 Fluorescence Polarisation Immunoassay 32
ix
1.6.4 Detection of analytes by immunoassay using up-converting phosphor
technology 32
1.6.5 Agglutination 33
1.6.6 Biosensors 33
1.7 Commercials Tests 36
1.8 Summary of Introduction 40
1.9 Aims of Thesis 41
Chapter 2
Materials & Methods 43
2.1 Materials 44
2.2 Equipment 46
2.3 Consumables 47
2.4 Standard Solutions 48
2.4.1 Cell culture media 48
2.4.2. SDS PAGE Solutions 49
2.5 Production of drug-protein conjugates 50
2.5.1 Conjugation of morphine-3-glucuronide to protein 50
2.5.2 Conjugation of cocaine to protein 50
2.5.3 Commercial conjugates 51
2.6 Immunisations for polyclonal and monoclonal
antibody production 51
2.6.1 Immunisation protocol for the production of monoclonal antibodies 51
2.6.2. Immunisation protocol for the production of monoclonal antibodies 52
2.6.3 Preparation of rabbit serum 53
2.6.4 Preparation of mouse serum 53
2.7 Production of monoclonal antibodies 53
2.7.1 Cell lines 53
2.7.2 Immunisation schedule 54
2.7.3 Fusion 54
2.7.4 Screening of hybridoma supernatants 55