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Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2017 Immunodetection of Porcine Blood in Foods Xingyi Jiang Follow this and additional works at the DigiNole: FSU's Digital Repository. For more information, please contact [email protected] FLORIDA STATE UNIVERSITY COLLEGE OF HUMAN SCIENCES IMMUNODETECTION OF PORCINE BLOOD IN FOODS By XINGYI JIANG A Thesis submitted to the Department of Nutrition, Food and Exercise Sciences in partial fulfillment of the requirements for the degree of Master of Science 2017 Xingyi Jiang defended this thesis on March 27, 2017. The members of the supervisory committee were: Qinchun Rao Professor Directing Thesis Shridhar K. Sathe Committee Member Timothy M. Logan Committee Member The Graduate School has verified and approved the above-named committee members, and certifies that the thesis has been approved in accordance with university requirements. ii ACKNOWLEDGMENTS Firstly, I would like to express my sincere gratitude to my major professor Dr. Qinchun Rao for the continuous support of my master study. I could not finish the thesis without him. As my major professor, Dr. Rao taught me all the knowledge and lab techniques. I am very grateful for giving me research assistantship in my master study. I also deeply thank Dr. Timothy Logan and Dr. Shridhar Sathe who served as my committee members. They gave me valuable advice when I was conducting my thesis. Secondly, I want to thank the entire faculty and staff at College of Human Sciences. They are all very helpful. I sincerely thank the Department of Nutrition, Food and Exercise Sciences for providing the opportunity and financial support to my master study. Particularly, I am grateful for Dr. Shridhar K. Sathe, Dr. Jeong-Su Kim and Dr. Gloria Salazar for letting me use the equipment in their labs. Also, I also thank all the lab mates, Behnam Keshavarz who helped me a lot during researches, Han Mu, Yuyun Wu and Mustafa Samiwala who were always willing support me. Finally, I would like to show my deepest gratitude to my family members including my parents, Jianhua Jiang and Jing Wang, grandparents, Zhichun Wang and Guiying Xu, my cousin, Judy Cai. They are supportive of every decision I have made. Without their understanding, I could not get through the difficulties. Also, my friends, although we are not even in the same time zone, they always show up immediately whenever I need them. I believe I will never come to his point without the help of these supportive people. iii TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... vi LIST OF FIGURES ...................................................................................................................... vii ABSTRACT ................................................................................................................................... ix CHAPTER 1 INTRODUCTION .....................................................................................................1 1.1 Background ...........................................................................................................................1 1.2 Rationale and Significance ....................................................................................................2 1.2.1 Rationale 1: The importance of developing immunoassays for porcine blood detection .................................................................................................................................3 1.2.2 Rationale 2: The importance of developing and characterizing different antibodies....5 1.2.3 Rationale 3: The importance of studying the stability of target analytes ......................6 1.3 Objectives and Hypothesis ....................................................................................................7 1.3.1 Objective 1: To characterize three antibodies ...............................................................7 1.3.2 Objective 2: To develop an icELISA for porcine blood detection via porcine hemoglobin .............................................................................................................................8 1.3.3 Objective 3: To improve a reported sELISA for porcine blood detection via plasma protein .....................................................................................................................................9 CHAPTER 2 REVIEW OF LITERATURE ..................................................................................10 2.1 Porcine Blood ......................................................................................................................10 2.1.1 Porcine hemoglobin .....................................................................................................11 2.2 Porcine Blood Application ..................................................................................................15 2.2.1 Animal feed supplement ..............................................................................................16 2.2.2 Emulsifier, binder, and gelling agent ..........................................................................17 2.2.3 Nutritional fortification ...............................................................................................19 2.2.4 Natural color enhancer ................................................................................................20 2.2.5 Bioactive compounds ..................................................................................................21 2.3 Needs for Porcine Blood Detection ....................................................................................22 2.3.1 Religious issues ...........................................................................................................23 2.3.2 Food safety concerns: Microbial safety ......................................................................25 2.3.3 Food safety concerns: Diseased pork ..........................................................................26 2.3.4 Food quality concerns: Fraudulent cases ....................................................................27 2.3.5 Health and environmental concerns ............................................................................28 2.4 Porcine Blood Detection Methods ......................................................................................29 2.4.1 Chromatography ..........................................................................................................29 2.4.2 Polymerase chain reaction (PCR)................................................................................30 2.4.3 Spectrophotometry ......................................................................................................31 2.4.4 Immunoassay ...............................................................................................................32 CHAPTER 3 INDIRECT COMPETITIVE ELISA FOR THE DETECTION OF PORCINE BLOOD IN FOODS ......................................................................................................................34 3.1 Materials ..............................................................................................................................34 3.2 Methods ...............................................................................................................................36 3.2.1 Production of mAbs ....................................................................................................36 3.2.2 Protein extracts preparation .........................................................................................38 3.2.3 Indirect non-competitive ELISA (inELISA) ...............................................................39 3.2.4 SDS-PAGE and Western blot .....................................................................................40 iv 3.2.5 Aqueous two-phase system (ATPS) ............................................................................42 3.2.6 Gel filtration chromatography .....................................................................................42 3.2.7 Gradient gel electrophoresis ........................................................................................43 3.2.8 Two-dimensional gel electrophoresis ..........................................................................43 3.2.9 icELISA .......................................................................................................................44 3.2.10 Data analysis .............................................................................................................45 3.3 Results and Discussion ........................................................................................................45 3.3.1 Antibody screening .....................................................................................................45 3.3.2 Antibody purification ..................................................................................................46 3.3.3 Antibody characterization ...........................................................................................46 3.3.4 Immunoaffinity ............................................................................................................50 3.3.5 Hemoglobin stability ...................................................................................................54 3.3.6 Assay development .....................................................................................................59
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