Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2004 Effect of Food Matrix on Amandin (an Allergen in Almond) Recovery and Immunorecognition Rashmi S. Tiwari Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF HUMAN SCIENCES Effect of Food Matrix on Amandin (an allergen in almond) Recovery and Immunorecognition By RASHMI S. TIWARI 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. Degree Awarded: Spring Semester, 2004 The members of the committee approve the thesis of Rashmi. S. Tiwari defended on 8th April 2004. Shridhar K. Sathe Professor Directing Thesis Kenneth H. Roux Committee Member Cathy W. Levenson Committee Member Approved: James M. Overton, Chair, Department of Nutrition Food, and Exercise Sciences Penny A. Ralston, Dean, College of Human Science The office of Graduate studies has verified and approved the above named committee members. ii This Master’s Thesis is dedicated to my parents –Tiwari, Satyanarayan and Tiwari, Naina, who, in not consciously trying to mold in there own image, have influenced me for more than they will ever know. They found no sacrifice too great to have me educated to this level. I also dedicate my achievement to my sisters and brother. Their love, support and encouragement provided me courage throughout. Thank you! Mom and Papa! iii ACKNOWLEDGEMENTS The completion of this thesis represents the contribution of many persons. I am indebted to my committee members, friends, and family, whose joint efforts on my behalf intersect within these pages. A thesis is created from the brooding, foreboding, and fearing of a student and the cajoling, affirming, and critiquing of a committee. Dr. Shridhar. K. Sathe my major professor provided healthy skepticism about the theory and the unconscious, but consistently affirmed the worthiness of this study. His comments influenced the study from a rough hunch to a finished product. He gave me clear and manageable goals, and was always incisive in his comments. My sincere thanks to him for all his personal and professional help. I deeply acknowledge Dr. Kenneth. H. Roux and Dr. Cathy Levenson for serving on my committee. I am also grateful to Dr. J. Michael Overton and Dr. Moffatt for their professional advice and support. I am also thankful to Dr. Bonnie Greenwood for providing me financial support through Resource and Technology center. Last but not the least I am also thankful to my fellow graduate students Mahesh, Harshal, Susan and Erin who shared the trials of graduate work and the frustrations of research, I extend current thanks for the friendship, especially to Mahesh. Things couldn’t have being that easier without them. My special thanks to Mengna Su. My family members mom, dad, sisters, brother and my relatives have always been the joy of my life. I’d like to take this opportunity to express my love to them and complete this acknowledgement. My special thanks to Dr. Mary Ann Moore an educator, counselor and friend who was always available for direction, support and encouragement. iv TABLE OF CONTENTS List of tables……………………………………………………………………………..…….viii List of figures…………………………………………………………………….………………xi Abbreviations…………….………………………………………………………….…….……xii Abstract………………………………………………………..…………………………….....xiii CHAPTER I Introduction………………………………………………………………………..……….……...1 Statement of the problem…………………………………………………….………...…...……..7 Research hypothesis…………………………………………………..……..………….…….…..8 Significance of the study…………………………………………………….……………….…..10 Limitation of the study………………………………………………………...………….……...11 CHAPTER II Review of the literature………………………………………………………………...……...…12 Detection of allergens……………………………………………………….……...……12 Immunoassays…………………………………………………………………...………13 Applications of immunoassays……………………………………….……...………..…14 Cross-reactivity……………………………………………………………………….….16 v Recovery studies…………………………………………………………………..……..17 CHAPTER III Materials…………………………………………………………………………………………20 Methods………………………………………………………………………….……………….25 Preparation of defatted food matrix samples……………………………………….....…26 Preparation of amandin………………………………………………………………..…26 Protein extractions ………………………………………………………………...…….26 Protein determination……………………………………………………….…………....26 Enzyme-linked immunosorbent assay (ELISA)……………………….…………….…..27 Electrophoresis and western blotting…………………………………………..………...27 Dot blots……………………………………………………………………………….…28 Dark chocolate experiment………………………………………………………………28 Effect of extraction step………………………………………………………………….29 Effect of extraction ratio…………………………………………………………………29 Effect of extraction buffer……………………………………………………………….29 Effect of pH……………………………………………………………………………..29 Effect of spiking almond flour…………………………………………..……………….30 vi Statistical analysis………………………………………………………………..………30 CHAPTER IV Results……………………………………………………………………………………………31 ELISA………………………………………………………………………….………...31 Western blotting…………………………………………………………….…...….……48 Cross-reactivity……………………………………………………………..……..……..48 Qualitative detection of amandin……………………………………………………..….58 Dot blotting………………………………………………………………………...……63 LOWRY………………………………………………………………………….………65 CHAPTER V Discussions……………………………………………………………………..….…….………88 Conclusions………………………………………………………………………….……….…..93 Appendix………………………………………………………………………………….…..….94 References………………………………………………………………………...……….……124 Biographical sketch………………………………………………………………..……..…..…131 vii LIST OF TABLES 1. Examples of food allergens homologous to pathogenesis related proteins…………….....2 2. Selected ELISA applications for the detection of food allergens………..………….…...18 3. List of food matrices…………………………………………………………………..…21 4. Experimental design…………………………………………………………..…………25 5. Detection of amandin in 100 mg of food samples spiked with different levels of amandin………………………………………………………………………...……..….32 6. The following tables shows the LSD values (p = 0.05) for comparing the means of recoveries within the category…………………………..…...…………………..………37 7. Category effect on amandin detection………………………………….…..………....…39 8. The following tables list the categories that are significantly different from each other at three spike levels…………………………………………………………………….…..40 9. The ratio of IC values of amandin (spike level 100 µg/100 mg of food)…………..…..42 50 10. The regression equation used for molecular weight determination……..……………….48 11. The molecular weights (kDa) of the protein bands identified in western blots (pAb)………………………….……………………………………………………..…...54 12. Amandin detection using Lowry assay…..……………………………………………....65 13. List of cross-reactive food matrices……………………………………………………...70 14. Detection of amandin (almond major protein) in dark chocolate (100 mg) at different spike levels..……………………………………………………………………………...74 15. The ratio of IC of amandin in dark chocolate…………………………..……………...75 50 16. Effect of spiking amandin (100 µg) before and after extraction on recovery…………………………………………………………………………………..76 17. The ratio of IC values of amandin when spiked either before or after 50 extraction……………………………………………………………………...………….77 18. Effect of ratio (food matrix: extraction buffer) on amandin (100 µg spike level) recovery. The two ratios tested are 1:10 and 1:20……………………………………………...…..79 19. The ratio of IC values of amandin extracted with different extraction 50 ratios……………………………………………………………………………………..80 20. Recovery of amandin (at 100 µg spike level) from different extraction buffers…………………………………………….………………………………….…..81 21. The ratio of IC values of amandin when spiked in different buffers………………..…82 50 viii 22. Recovery of amandin from food matrices in different buffers (spike level= 100 µg)………………………………………………………………………………………..82 23. The IC values of amandin from food matrices in different buffers (spike level= 100 50 µg)……………………………………………………………………………….…….…83 24. Recovery of amandin from BSB (pH of 0.1 M BSB adjusted with 1 M HCl)………..…85 25. The ratio of IC values of amandin in BSB (pH adjusted with 0.1M HCl)…...………...85 50 26. Effect of pH on amandin detection……………………………………………..………..86 27. Detection of amandin in foods spiked with almond flour…….……………………….…87 28. One-Way ANOVA for detection of amandin in 100 mg of food samples spiked with different levels of amandin…………………………………………...…..………….…..94 29. One-way ANOVA for comparing mean within the category of food matrices…………………………………………………………………….….……….103 30. One-Way ANOVA for detection of amandin in 100 mg spiked foods to compare the recoveries across the food matrices……………………………………………….…...107 31. One-Way ANOVA for detection of amandin in different food categories spiked with different levels of amandin………………………...……………………………...……108 32. One-Way ANOVA for detection of amandin to compare the recoveries across the food categories……………………………………………………………………………….109 33. One-Way ANOVA for detection of amandin in dark chocolate (100 mg) at different spike………………………………………………………………………………….…109 34. One-Way ANOVA for recovery of amandin (at 100 µg spike level) from different extraction buffers……………………………………………………………………….109 35. One-Way ANOVA for recovery of amandin from BSB (pH of 0.1 M BSB adjusted with 1 M HCl).……………………………..…………………………………………...……109 36. One-Way ANOVA for effect of spiking amandin (100 µg) before and after extraction on recovery…………………………………………………………………………….…..110 37. One-Way ANOVA for effect of ratio (food matrix: extraction buffer) on amandin (100 µg spike level) recovery. The two ratios tested are 1:10 and 1:20………………………………………………………………………….………….111 38. One-Way ANOVA for recovery of amandin from food matrices in different buffers (spike level-100 µg)…………………………………………………………………….112 39. One-Way ANOVA for recovery of amandin from food matrices using Lowry assay (spike level-100 µg)……………………..……………………………………..……….112 40. One-way ANOVA for IC values……………………………………………………...113 50 41. One-way ANOVA for IC values……………………………………..……………….120 50 ix
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