Development of a Specific Enzyme Linked Immunosorbent Assay (ELISA) for the Detection of Fluoroquinolone Antibiotic Residues in Chicken Liver, Prawn and Milk Muhammad Zahid Thesis submitted in partial fulfilment of the requirement for the Degree of Master of Science (Research) School of Chemical Engineering The University of New South Wales April, 2011 ABSTRACT Extensive utilisation of fluoroquinolones (FQs) in agricultural and aquacultural practices leads to two major food safety issues: 1) the issues regarding the presence of FQs residues in food and 2) the development of FQs resistant bacteria in animals, which may be transferable to humans. This may have an implication to human health, in particular for the treatment of infection. This thesis describes the design and synthesis of novel haptens for (enrofloxacin) ENR, ciprofloxacin and norfloxacin, the production of specific antibodies, and the formatting and characterizing of an indirect competitive Enzyme‐Linked ImmunoSorbent Assay (ELISA) for detection of ENR. The design and synthesis of FQs haptens involved the following approaches: 1) synthesising ENR hapten by attaching a tert‐butyl linker on a carboxylic group, and 2) synthesising ciprofloxacin and norfloxacin haptens by attaching a 4‐ bromobutane NHS ester and bromocrotyl NHS ester linkers respectively on the piperazinyl moiety. Highly specific polyclonal antibodies were generated against the ENR‐Keyhole Limpet Haemocyanine (KLH) conjugate. The optimized ELISA exhibited higher sensitivity in a homologous assay than a heterologous assay, suggesting the developed antibody was ‐1 extremely specific to ENR. The ELISA displayed an IC50 value of 11.7 µg L ± 1.7 with a limit of detection (LOD) value of 2.4 µg L‐1 ± 0.4. High specificity of the developed assay was evidenced by low cross‐reactivity to seven structurally related FQs compounds (danofloxacin, enofloxacin, sarafloxacin, perfloxacin, nalidixic acid, ciprofloxacin and norfloxacin). The effects of surfactants (Tween 20), water miscible organic solvent (methanol, ethanol, acetonitrile, and acetone) and pH conditions (5.5‐9.5) were also evaluated. Briefly, Tween 20 affected considerably on colour development, but not the assay sensitivity. Of the solvents tested, up to 5% methanol showed no significant effects on the assay sensitivity. The sample preparation were also optimized for milk, chicken liver and prawn, yielding the recoveries between 64 (± 3) and 125 (± 8)%. This ELISA will be particularly useful for screening ENR residues in animal and marine derived products to improve antibiotic safety in developing countries such as Indonesia. i ACKNOWLEDGEMENTS “In the name of ALLAH, the most gracious and merciful” First and foremost I would like to express my sincerest gratitude to ALLAH SWT, my Lord and Cherisher, for guiding and blessing in every single step of my life. Indeed, without his help and will, nothing is accomplished. I am heartily thankful to my supervisor, Dr. Nanju Alice Lee, whose encouragement, guidance and support from the very early to the final stage of this research, enabled me to develop an understanding of the subject as well as gave me extraordinary experiences throughout the work. Her encouragement has triggered and nourished my intellectual maturity that I will benefit from. I am grateful in every possible way and hope to keep up our collaboration in the future. I gratefully acknowledge my co‐supervisor, A/Prof. Naresh Kumar for his advice, expertise, and supervision. It has been an honour to have had the opportunity to work in his laboratory. I also would like to express my gratitude to Dr. George Iskander for his involvement, ideas, research passion and crucial contribution has made him one of the backbones of this research. Many thanks go to Dr. Victor Wong for the precious time rendered in proofreading this thesis, including the critical comments and scientific ideas forwarded. I would like to thank Camillo Toraborelli for his technical assistance in the laboratory and his kindness in putting every requested chemical on my bench. Special thanks also to all fellow researchers in the Food Science and Nutrition Research laboratory, School of Chemical Engineering; Eriyanto Yusnawan, Maria Veronica Hoie, Karrie Kam, Yang Lu, Kim‐Yen Phan‐Thien, Ebtihal Khodijah, Chatchaporn Uraipong and Norma Karim, as well as those who are affiliated with, the Organic Chemistry laboratory, School of Chemistry; Samuel Kutty, Hakan Kandemir, Kitty Ho, Ren Chen, Rick Zhang, Raymond Chen, Adeline Lukmantara and Asep Kurnia Permana, for their support, knowledge, sharing, laughs, and even tears. I am so grateful to have you guys who are always around. You are such wonderful people and always make our laboratory such an incredible place to be. ii For financial assistance, I would like to express my deepest gratitude to AusAID through Australian Development Scholarship (ADS) for giving me a great opportunity to study at The University of New South Wales, Australia. Without this support, this research project would have been impossible. I would like to record my gratitude to the Indonesian government, and more specifically, The National Veterinary Drug Assay Laboratory, The Ministry of Agriculture of Republic of Indonesia, for allowing me to improve my skills and knowledge through this research project in Australia. This research project would not have been possible without the support of numerous other people, so I offer my regards and blessings to all who supported me in any way for the duration of the project. Lastly, I would like to dedicate this thesis to my beloved families and especially for my beloved wife; Isnindar, my little angel; Maura Thalita Chairinniswa, for their prayers, patience, understanding and endless love, throughout the duration of my studies. iii ABBREVIATIONS Ab‐ENR1 polyclonal antibody of ENR Amax maximum absorbance APCI atmospheric pressure chemical ionization BSA bovine serum albumin cBSA cationised‐bovine serum albumin CBT checkerboard titration CDCl3 deuterated chloroform CIP ciprofloxacin a conjugate of ciprofloxacin N‐hydroxysuccinimide ester and CIP1‐OA ovalbumin a conjugate of ciprofloxacin butyl N‐hydroxysuccinimide ester and CIP2‐OA ovalbumin 1‐cyclohexyl‐3‐(2‐morphplinyl‐4‐ethyl) carbodiimide methyl p‐ CMC toluene sulfonate CNS central nervous system CV coefficient of variation D2O deuterium oxide DAD diode array detection DAN danofloxacin DCC dicyclohexylcarbodiimide DCM dichloromethane DEPT distortionless enhancement by polarization transfer DIC diisopropylcarbodiimide DMAP 4‐dimethylaminopyridine DMF dimethylformamide DMSO dimethylsulfoxide DNA deoxyribonucleic acid EDC 1‐ethyl‐3‐(3‐dimethyl‐aminopropyl) carbodiimide hydrochloride iv ELISA enzyme linked immunosorbent assay ENO enoxacin ENR enrofloxacin a conjugate of ENR N‐hydroxysuccinimide ester and keyhole limpet ENR1‐KLH haemocyanin ENR1‐OA a conjugate of ENR N‐hydroxysuccinimide ester and ovalbumin ENR2‐OA conjugate of ENR acid and keyhole limpet haemocyanin ESI electrospray ionization EtOH ethanol EU European Union FAO/WHO Food and Agriculture Organisation/World Health Organisation FCL full cream liquid milk FCP full cream milk powder FG fish gelatine FLD fluorescence detection FLU flumequine FQs fluoroquinolones FSANZ Food Standard Australia New Zealand GAT gatifloxacin GC/MS gas chromatography/mass spectrometry GI gastrointestinal HAS human albumin serum HPLC high performance liquid chromatography HRMS high resolution mass spectrometry HRP horseradish peroxidise IBCF isobutylchloroformate Ig immunoglobulin IgG immunoglobulin g JEFCA joint expert committee on food additives v KLH keyhole limpet haemocyanin LC/MS liquid chromatography mass spectrometry LC/MS‐MS liquid chromatography/tandem mass spectrometry LOD limit of detection LOQ limit of quantification LRMS low resolution mass spectrometry Lv1 chicken liver from an organic source Lv2 chicken liver from Coles supermarket Lv3 chicken liver from butcher MAb monoclonal antibody MAR marbofloxacin MeOH methanol MRLs maximum residue limits NaH sodium hydride NAL nalidixic acid NAL‐OA a conjugate of nalidixic acid and ovalbumin NHS N‐hydroxysuccinimide NMR nuclear magnetic resonance NOR norfloxacin NOR‐OA a conjugate of norfloxacin and ovalbumin NSAID non steroid anti inflammatory drug OFL ofloxacin OA ovalbumin OXO oxolinic acid PAb polyclonal antibody PBS phosphate buffer saline PEF pefloxacin PEF‐OA a conjugate of pefloxacin ovalbumin vi Pr1 local prawn Pr2 Thai prawn Pr3 Malaysian prawn Rf retardation factor RO reverse osmosis SAR sarafloxacin SAR‐OA a conjugate of sarafloxacin and ovalbumin SARs structure activity relationships SD standard deviation SKL skim milk liquid SKP skim milk powder TEA triethylamine TFA tetrafluoroacetic acid TLC thin layer chromatography TMB 3,3’,5,5’‐tetramethylbenzidine USP the U.S. of pharmacopeia UV ultraviolet vii TABLE OF CONTENTS ABSTRACT ....................................................................................................... I ACKNOWLEDGEMENTS ..................................................................................... II ABBREVIATIONS ............................................................................................. IV LIST OF FIGURES ........................................................................................... XIII LIST OF TABLES ............................................................................................. XX CHAPTER 1. INTRODUCTION .............................................................................