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Identification of Vibrio Anguillarum and Vibrio Ordalii by a Monoclonal Antibody Coagglutination Assay

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Identification of Vibrio Anguillarum and Vibrio Ordalii by a Monoclonal Antibody Coagglutination Assay Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1990 Identification of Vibrio Anguillarum and Vibrio Ordalii by a Monoclonal Antibody Coagglutination Assay. Zhongshi Wang Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Wang, Zhongshi, "Identification of Vibrio Anguillarum and Vibrio Ordalii by a Monoclonal Antibody Coagglutination Assay." (1990). LSU Historical Dissertations and Theses. 5024. https://digitalcommons.lsu.edu/gradschool_disstheses/5024 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. 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Order Number 9112271 Identification ofVibrio anguillarum and Vibrio ordalii by a monoclonal antibody coagglutination assay Wang, Zhongshi, Ph.D. The Louisiana State University and Agricultural and Mechanical Col., 1990 UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IDENTIFICATION OF VIBRIO ANGUILLARUM AND VIBRIO ORDALII BY A MONOCLONAL ANTIBODY COAGGLUTINATION ASSAY A dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Microbiology by Zhongshi Wang B.M., Beijing Medical University, 1983 Beijing, China Aug,1990 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION To my parents, brothers, and sisters. ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENT I would like to express thanks and sincere appreciation to Dr. R.J. Siebeling for giving me the opportunity to pursue graduate studies in Microbiology and for his patience, helpful direction, continuous support throughout the course of this research and the assistance in the preparation of this dissertation. I would like to express sincere appreciation to Dr. L.T. Hart, V.R. Srinivasan, E.C. Achberger and to my minor professor Dr. E.S. Younathan for countless examples of advice, support and patience. I also express sincere gratitude to the members of Dr. Siebeling's research group for their friendships. I would like to thank Cindy Henk for helping me to prepare the slides for my oral presentations. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents Page Dedication.......................................... ii Acknowledgement...... iii Table of Contents................................... iv List of Tables....................................... Abstract............................................ ix Introduction......................................... 1 Chapter I Production of Species Specific Anti-H Monoclonal Antibodies Against Vibrio anguillarum and Vibrio Ordalii......... 20 Introduction........................... 21 Materials and Methods.................. 29 Results and Discussion................. 47 Chapter II Serological Identification of Vibrio anguillarum and Vibrio ordalii by Using Lipopolysaccharide (0) Antigens......... 65 Introduction.................. 66 Materials and Methods................. 75 Resultes and Discussion................89 Literature Cited.................................. 131 Vita............................................. 147 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLE Table Chapter I. la. Vibrio anguillarum and Vibrio ordalii strains used in serological assays throught this study........................ 30 lb. Heterologous Vibrio species used in this study..................................... 32 2. Isotype of monoclonal antibodies reactive against flagellar core protein of V. anguillarum and V. ordalii....... 48 3. Serological reactivity of six anti-MAbs examined by ELISA, with flagellar core proteins prepared from 10-serogroups of V. anguillarum and V. ordalii strains (Larsen 0-scheme).......................... 50 4. H-antigen relationships among Vibrio fish pathogens, examined by ELISA with anti-V. anguillarum and anti-V. ordalii H monoclonal antibodies (MAbs)............. 52 5. Coagglutination patterns for two affinity-purified anti-V. ordalii flagella monoclonal antibodies. Antibodies eluted from protein-A Sepharose armed with 10% S.. aureus........................ 54 6a. Serological reactivity of anti-V. ordalii H MAbs examined by coagglutination reaction. Anti-H coagglutination reagent tested against 10 V. anguillarum O-serotypes obtained from Denmark...................... 56 6b. Serological reactivity of anti-V. ordalii H MAbs examined by coagglutination reaction. Anti-H coagglutination reagent tested against V. anguillarum isolated from taiwan................................ 57 6c. Serological reactivity of anti-V. ordalii H MAbs examined by coagglutination reaction. Anti-H coagglutination reagent tested against V. anguillaxrum isolated from Japan................................. 58 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6d. Serological reactivity of anti-V. ordalii H MAbs examined by coagglutination reaction. Anti-H coagglutination reagent tested against heterologous Vibrio fish pathogens or Vibrio species which might share H antigen determinants............................... 59 7. Serological survey by Vibrio anguillarum and Vibrio ordalii isolated diseased fishes or marine environment and other Vibrio species by coagglutination with reagents armed with anti-V. ordalii H MAbs..................... 61 Chapter II 1. A summary and comparision of five serotyping systems for V. anguillarum based on cell wall (0) associated lipopolysaccharide antigens: Correlation of 0-antigen nomenclature................................ 68 2. Origin and source of the bacterial strains used in this study.......................... 76 3. Schedule of immunization for preparation of rabbit antiserum...... 82 4. Direct slide agglutination (DSA) and passive hemagglutination (PHA) titers for rabbit anti-0 sera produced against each of the 10 Danish V. anguillarum O-serotypes and tested against vaccine strains........... 91 5. Agglutination patterns for 10 anti-V. anguillarum sera raised in rabbits immunized with heat-killed whole cell vaccines and tested against homologous vaccine strains and heterologous vaccine strains.................................... 92 6a. Agglutination patterns for 10 different anti- V. anguillarum sera raised in rabbits immunized with heat-killed whole cell vaccines and tested against V. anguillarum strains isolated from Taiwan........................ 94 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6b. Agglutination patterns for 10 different anti- V. anguillarum sera raised in rabbits immunized with heat-killed whole cell vaccines and tested against V. anguillarum strains isolated from Japan............ 95 6c. Agglutination patterns for 10 different anti- V. anguillarum sera raised in rabbits immunized with heat-killed whole cell vaccines and tested against V. anguillarum strains isolated from America...................... 97 6d. Serological examination of other than V. anguillarum and V. ordalii vibrios isolates by direct-immune
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