Development of Immunoassays for the Detection of 2-Methylisoborneol and Monensin in Water Samples by Rashidah Sukor A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Food Science Guelph, Ontario, Canada © Rashidah Sukor, July, 2013 ABSTRACT DEVELOPMENT OF IMMUNOASSAYS FOR THE DEVELOPMENT OF 2- METHYLISOBORNEOL AND MONENSIN IN WATER SAMPLES Rashidah Sukor Advisor: University of Guelph, 2013 Professor J. Christopher Hall Immunoassays for 2-methylisoborneol (MIB) and monensin in water were developed, devised and tested to see if the sensitivity could be established and improved. MIB and monensin are hydrophobic haptens with molecular weights of 168 and 671 Da, respectively. Rabbits were immunized with (-) camphor-BSA and (-) borneol-BSA for the production of polyclonal antibodies (pAbs) to MIB. Monoclonal antibodies (mAbs) were produced in Mus musculus using (-) camphor-BSA as immunogen. (+) Bornylamine-thyroglobulin (TG) and MIB-TG were synthesized and used as plate coatings. For the monensin immunoassay, monensin was conjugated to BSA and OVA for immunogen and plate coating, respectively. Several physical parameters that affect the sensitivity of immunoassays including pre-incubation of antibody and antigen, incubation time and temperature, detergent, organic solvents, and ionic strength were evaluated. Improvement of immunoassay sensitivity was also performed by reducing the concentrations of coating antigen and antibodies and using alternative reporter systems such as chemiluminescence (CICL-ELISA), tyramide signal amplification (TSA) and biotin-streptavidin. Different assay formats, i.e., competitive indirect and competitive direct were also compared. Usability of both pAb-based immunoassays for MIB and monensin was evaluated in fortified water samples. A polyclonal-based (pAb) ELISA for MIB had a detection limit of 4.8 ng mL-1 and -1 an IC50 of 105 ng mL . Rabbits immunized with (-) camphor-BSA showed a higher immune response than rabbits immunized with (-) borneol-BSA. One clone (i.e., 4F11) of fourteen characterized clones was used to create the monoclonal antibody (mAb)-based -1 -1 ELISA, which had an IC50 of 100.2 ng mL and an LOD of 1.9 ng mL . The pAb- and mAb-based CI-ELISA were not specific to MIB alone and cross reacted with camphor and camphor-like compounds. Meanwhile, a pAb-based ELISA for monensin produced a -1 -1 detection limit of 0.1 ng mL and had an IC50 of 1.056-1.090 ng mL with high specificity to monensin. Other reporter systems did not improve the sensitivity of the immunoassays significantly. MIB and monensin polyclonal-based assays showed good correlation to analytical instrumental methods (i.e., GC-MS and LC-MS) in fortified water samples. With a detection limit of ca. 5 ng mL-1 and 0.1 ng mL-1 for MIB and monensin, respectively, both polyclonal-based assays can be used for detection of these analytes in water from different sources and employed as screening tools to complement GC/HPLC-MS instrument methods. Keywords: immunoassay, polyclonal antibodies, monoclonal antibodies, 2- methylisoborneol, monensin PREFACE This thesis (Chapter 3, 4 and 5) is organized as manuscript to be submitted for publication in peer reviewed journals. Hence, a repetition of some information is unavoidable. All chapters were written by Rashidah Sukor as primary author and J. C. Hall as corresponding author. Editorial comments in Chapter 3 were contributed by Dr. J.C. Hall and Gabrielle Richard; Chapter 4 by Dr. J.C. Hall and Dr. Jyothi Kumaran and Chapter 5 by Dr. J.C. Hall, Gabrielle Richard and Shokouh Makvandi-Nejad. The Chapters are listed below: Chapter 3: Sukor, R., Hall, J.C. 2013. Development of a polyclonal-based immunoassay for the detection of 2-methylisoborneol in water. (To be submitted to Journal of Agricultural and Food Chemistry) Chapter 4: Sukor, R., Hall, J.C. 2013. Development of monoclonal-based immunoassay for the detection of 2-methylisoborneol. Chapter 5: Sukor, R., Makvandi-Nejad, S., Veldhuis, L., Hall, J.C. 2013. Development and comparison of a chemiluminescence and direct immunoassays for the detection of monensin. (To be submitted to Journal of Environmental Quality) iv ACKNOWLEDGEMENTS First and foremost, Alhamdulillah, All Praise belongs to Allah, the Almighty, whom has bestowed me the many blessings in life including the opportunity to undertake and challenge myself to pursue a PhD degree, without which nothing is possible. This dissertation would not have been conceivable without the guidance and assistance of many individuals who in one way or another contributed and extended their helping hand in the preparation and completion of this study. Utmost gratitude to my advisor, Dr. Chris Hall, whom I shared valuable knowledge and resources that have guided me throughout my PhD program. Thanks Chris for your encouragement, for believing in me in those difficult times. You have taught me the meaning of perseverance. Special thanks to Dr. Mansel Griffiths, Dr. Keith Solomon and Dr. Mark Hewitt for being on my advisory committee and constructive criticism during the preparation of the thesis. I am grateful to the members of my examination committee, Dr. Lamia L. Hocine, Dr. Keith Warriner, and Dr. Rickey Yada for their time and insightful comments. I would like to express a great appreciation to members of Dr. Hall lab from 2007-2013 especially to Ted, John, Zheng, Ju, and Filomena, for their invaluable friendships; Gabrielle for always being there through the hardship and for being my outstanding thesis editor; Xaver for his friendship and sharing ELISA technical tips and tricks; Yong Qing and Xue Mei for the life experience sharing and Shokouh for her friendship and critiques in the monensin chapter. In addition, a deepest gratitude to Lucy for her help and guidance during preparation of tissue-culture works and Sushma for her assistance in the validation work. Furthermore, I would also like to acknowledge Dr. Fernando Rubio (Abraxis LLC) for providing the conjugates and to Teri Otto (ImmunoPrecise) for her technical assistance. v I am grateful to Ministry of Higher Education of Malaysia and Universiti Putra Malaysia for the financial support throughout my doctoral study. Special thanks to Malaysian students and families especially Haniff and Azizah for sharing their humble abode and providing the warmth of a family and to others, especially Nik Haniza, Ahmad Peter, Madzlan, Siti, Irill, Izaty, and Sabri for their support and the endless tastes of home. Not forgetting Raj, Jiap, and my roommates whose friendship I will always cherish and Government of Malaysia for the financial support. Deepest gratitude to my family for their endless support, sacrifice and unconditional love throughout the entire process which have helped keep my sanity intact at most times, especially to my mom for caring of my daughter in my absence, "Terima kasih, Mak’’. Sincere gratitude also goes to my late father who had taught me the value and meaning of life. And last, but not least, I dedicate this thesis to the love of my life, Mia Hana, for being the continuous motivation. vi TABLE OF CONTENTS Acknowledgements ...................................................................................................... v Table of Contents ........................................................................................................vii List of Tables ............................................................................................................. xiii List of Figures .............................................................................................................xv List of Abbreviations ............................................................................................... xviii 1 GENERAL INTRODUCTION AND RESEARCH OBJECTIVES ......................... 1 2.1 Introduction .............................................................................................. 1 1.2 Research objectives ................................................................................ 4 2 LITERATURE REVIEW ..................................................................................... 6 2.1 Introduction .............................................................................................. 6 2.2 Production of antibody ............................................................................ 8 2.2.1 Basic antibody structure ................................................................. 8 2.2.2 Generation of an immune response ................................................ 9 2.2.3 Polyclonal antibodies .....................................................................10 2.2.4 Monoclonal antibodies ...................................................................11 2.2.5 Anti-hapten antibodies ...................................................................13 2.2.6 Kinetics of antibody-antigen interaction ..........................................14 2.3 Enzyme immunoassay ............................................................................15 2.3.1 Immunoassay format ....................................................................20 2.3.1.1 Direct competitive ELISA .........................................................20 2.3.1.2 Indirect competitive ELISA .......................................................21 2.3.1.3 Sandwich ELISA .......................................................................22 2.3.2 Heterogenous and homogenous assays .......................................22