Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2008 Characterization of Monoclonal Antibody Specific to Fish Major Allergen Parvalbumin Kamil Gajewski Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] FLORIDA STATE UNIVERSITY COLLEGE OF HUMAN SCIENCES CHARACTERIZATION OF MONOCLONAL ANTIBODY SPECIFIC TO FISH MAJOR ALLERGEN – PARVALBUMIN By KAMIL GAJEWSKI 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: Summer Semester, 2008 The members of the Committee approve the Thesis of Kamil Gajewski defended on June 23, 2008. ______________________________ Yun-Hwa Peggy Hsieh Professor Directing Thesis ______________________________ Kenneth H. Roux Outside Committee Member ______________________________ Cathy W. Levenson Committee Member Approved: ______________________________________________________________________ Bahram H. Arjmandi, Chair, Department of Nutrition, Food and Exercise Sciences ___________________________________________ Billie J. Collier, Dean, College of Human Sciences The Office of Graduate Studies has verified and approved the above named committee members. ii ACKNOWLEDGEMENTS I am grateful for all the support I have received during preparation of this thesis. I especially would like to thank my advisor, Dr. Yun-Hwa Peggy Hsieh, for insightful conversations during the development of the ideas in this thesis, for helpful comments on the text, and for keeping me focused in my research. Without her encouragement and contribution this work would not have been completed. I would also like to thank Dr. Cathy Levenson and Dr. Kenneth Roux for agreeing to be on my thesis committee despite their extremely busy schedule. I want to thank all of my colleagues in Dr. Hsieh’s laboratory for the support, advice, and friendship. Qinchun Rao, Jack Ofori, Yi-Tien Chen and Marsha Fridie made the laboratory a wonderful place to work. Special thanks to my mom and uncle who supported my dreams and aspirations. Finally, I also want to thank my wife, Iwona, for her patience and for helping me keep my life in proper perspective and balance. Her faith in me helped me to overcome all difficulties. iii TABLE OF CONTENTS List of Tables ...................................................................................................... vi List of Figures ...................................................................................................... vii Abstract ............................................................................................................... viii 1. Introduction .................................................................................................... 1 2. Literature Review............................................................................................ 4 2.1 Fish Allergy ....................................................................................... 4 2.2 Major Fish Allergen - Parvalbumin ................................................... 5 2.3 Other Fish Allergens .......................................................................... 6 2.4 Fish Allergen Contamination ............................................................. 7 2.5 The Food Allergen Labeling and Consumer Protection Act ............. 9 2.6 Allergen Detection Methods .............................................................. 10 2.6.1 Allergen protein-based detection methods ............................. 10 2.6.1 Allergen DNA-based detection methods ............................... 12 3. Hypotheses and Objectives ............................................................................. 14 3.1 Hypotheses ......................................................................................... 14 3.2 Objectives .......................................................................................... 14 4. Materials and Methods .................................................................................... 15 4.1 Materials ............................................................................................ 15 4.2 Protein extraction from fish and meat samples .................................. 16 4.3 Indirect ELISA ................................................................................... 16 4.4 Ca2+ dependent binding assay ............................................................ 17 4.4 Epitop comparison ............................................................................. 17 4.6 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western blot .................................................................. 18 5. Results and Discussion ................................................................................... 19 5.1 Specificity of MAbs 3E1 and PARV-19 ............................................ 19 5.2 Thermal-stable protein profile in cooked fish extracts ...................... 20 5.3 Antigenic protein for MAb 3E1 ......................................................... 21 5.4 Ca2+ sensitive epitopes ....................................................................... 22 5.5 Epitope comparison ........................................................................... 23 6. Conclusions ..................................................................................................... 25 iv APPENDICES .................................................................................................... 26 A Protocols and Procedures ...................................................................... 26 B Figures and Tables ................................................................................ 30 REFERENCES ................................................................................................... 38 BIOGRAPHICAL SKETCH .............................................................................. 45 v LIST OF TABLES Table 1: Immunoreactivity of MAbs 3E1 and PARV-19 against cooked shellfish, meat, poultry and food additives samples determined by indirect ELISA .................................................................................... 30 Table 2: Immunoreactivity of MAbs 3E1 and PARV-19 against cooked fish samples determined by indirect ELISA ............................................... 31 vi LIST OF FIGURES Figure 1: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) profiles of 20 cooked fish species .......................................... 32 Figure 2: Antigenic protein banding patterns in cooked fish extracts by Western blot analysis, using MAbs 3E1PARV-19 by Western blot analysis ................................................................................................ 33 Figure 3: Comparison of the antigenic protein banding patterns in cooked fish extracts using MAbs 3E1 and PARV-19 by Western blot ................. 34 Figure 4: Effect of presence and absence of calcium on the immunoreactivity of anti-parvalbumin specific MAbs 3E1 with fish and meat extracts determined by indirect ELISA ........................................................... 35 Figure 5: Effect of presence and absence of calcium on the immunoreactivity of anti-parvalbumin specific MAbs PARV-19 with fish and meat extracts determined by indirect ELISA .............................................. 36 Figure 6: Epitope comparison of MAbs 3E1 and PARV-19 .............................. 37 vii ABSTRACT Fish allergy is a worldwide problem, especially in industrialized countries where fish consumption is high. Fish contains a wide variety of proteins but, only few of them are responsible for triggering an allergic reaction. The major fish allergen, parvalbumin, is a low molecular weight (10-13 kD) heat-stable protein. High homology in amino acid sequences and antibody cross-reactivities have been demonstrated for parvalbumin in different fish species. Although several detection methods based on specific antibodies or DNA amplification are currently employed for detection of allergic components in food products, there is limited number of studies reporting methods for the detection of fish allergens. This study aimed to investigate the antigen binding characteristics of a monoclonal antibody (MAb) 3E1 by comparing its immunoreactivity against various fish and other animal species with a commercially available anti-frog parvalbumin monoclonal antibody (PARV-19) to evaluate the usefulness of MAb 3E1 as a anti-fish parvalbumin reagent. MAb 3E1 was previously developed in our laboratory against heat-treated catfish crude sarcoplasmic protein extract. The antigen binding characteristics of this antibody was investigated by comparing its immunoreactivity against soluble proteins extracts from various cooked fish and other animal meats with MAb PARV-19. Non-competitive indirect ELISA was performed to examine the immunoreactivity of both MAb 3E1 and MAb PARV-19 with sample extracts. Western blot was performed to compare the antigenic protein banding patterns in cooked fish extracts using these two MAbs. Results showed that MAb 3E1 cross reacted with majority of tested fish species and recognized a thermal-stable protein with a molecular weight range of parvalbumin in the extracts. Moreover, ELISA and western blot results revealed that both MAbs 3E1 and PARV-19 had almost identical reaction patterns to the fish species tested. The antigenic protein banding pattern in various