By - DORAS

[Pages:327]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 '0 0 .

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 o f the SMT Project Group from Envitec, Nunc, and University o f 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 methamphetaminebovine 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 BDB BEC BIA BSA BtG CDR CE CV DNA EDC EDTA EME ELISA EMIT Fab Fc FCS FPIA Fv GC/MS HAT HPLC HT HBS IgG IgA IgD IgE IgM

ka

ka

antigen presenting cell benzodioxole-5-butanamine benzoylecgonine biomolecular interaction analysis bovine serum albumin bovine thyroglobulin complementarity determining regions of antibody capillary electrophoresis coefficient o f variation deoxyribonucleic acid N-ethyl-N'-(dimethylaminopropyl) carbodiimide ethylenediaminetetra acetic acid ecgonine methyl ester enzyme-linked immunosorbent assay enzyme-multiplied immunoassay technique binding region of antibody above the hinge region constant region of antibody molecule foetal calf serum fluorescence polarisation immunoassay variable binding fragment of antibody gas chromatography/mass spectroscopy hypoxanthine aminopterin thymidine high performance liquid chromatography hypoxanthine thymidine Hepes buffered saline immunoglobulin class G immunoglobulin class A immunoglobulin class D immunoglobulin class E immunoglobulin class M equilibrium association affinity constant association rate constant

vi

Kd kd MAb MDA MDMA MBDB MDEA MW NEAA NIDA NHS OVA PAGE PBS PEG pH SAMHSA

scFv SE SDS SMT S/P SPR THC THC-COOH THY UV Vh Vl WHO

equilibrium dissociation affinity constant dissociation rate constant monoclonal antibody 3,4-methylenedioxyamphetamine 3,4-methylenedioxymethamphetamine 3,4-methylenedioxyphenyl-2-butanamine 3,4-methylenedioxy-N-ethylamphetamine molecular weight non-essential amino acids National Institute of Drugs of Abuse N-hydroxysuccinimide ovalbumin polyacrylamide gel electrophoresis phosphate buffer saline polyethylene glycol log of the hydrogen ion concentration Substance Abuse and Mental Health Administration single chain Fv antibody derivative standard error sodium dodecyl sulphate Standard, Measurements and Testing Framework saliva/plasma ratio surface plasmon resonance tetrahydrocannabinol tetrahydrocannabinol carboxylic acid thyroglobulin ultraviolet variable region of heavy chain variable region of light chain World Health Organisation

Service

vii

Units

?c cm g KDa Kg 1 Mg III pM M mg min ml mm nm nM mol rpm RU sec, s v/v w/v

degrees Celcius centimetres grams kilodaltons kilogram litre microgram microlitre micromolar molar miligram minute millilitre millimetres nanometre nanomolar molar revolutions per minute response units seconds volume per unit volume weight per unit volume

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