Production and purification of rabbit igG antibodies against fasciola gigantica excretory- secretory antigen.pdf

NGUYEN TAT THANH UNIVERSITY NGUYEN TAT THANH True learning, true practice, true success, true future FACULTY OF BIOTECHNOLOGY GRADUATION THESIS PRODUCTION AND PURIFICATION OF RABBIT IgG ANTIBODIES AGAINST Fasciola gigantica EXCRETORY/SECRETORY ANTIGEN Student’s name : Xuan-Thu Chau Student ID : 1411530308 Supervisor : Huu-Hung Nguyen, PhD Ho Chi Minh City, 2019 NGUYEN TAT THANH UNIVERSITY NGUYEN TAT THANH True learning, true practice, true success, true future FACULTY OF BIOTECHNOLOGY GRADUATION THESIS PRODUCTION AND PURIFICATION OF RABBIT IgG ANTIBODIES AGAINST Fasciola gigantica EXCRETORY/SECRETORY ANTIGEN Student’s name : Xuan-Thu Chau Student ID :1411530308 Major : Biotechnology Supervisor : Huu-Hung Nguyen, PhD Ho Chi Minh City, 2019 NGUYEN TAT THANH UNIVERSITY SOCIALIST REPUBLIC OF VIETNAM FACULTY OF BIOTECHNOLOGY Independence - Liberty - Happiness -oOo ASSIGNED TASK OF GRADUATION THESIS Full name: Xuan-Thu Chau Student ID: 1411530308 Major: Biotechnology Class: 14DSH02 Thesis’s title: Production and purification of rabbit IgG antibodies against Fasciola gigantica excretory/secretory antigen Objectives - Production of rabbit IgG antibodies against Fasciola gigantica excretory/secretory antigen Contents - Immunization of rabbit with excretory/secretory antigen obtained from adult worm of Fasciola gigantica - Purification of polyclonal IgG antibodies from the immunized animals - Assessment of bio-activity of purified IgG antibodies Execution time: from 02/2019 to 08/2019 Supervisor: Huu-Hung Nguyen, PhD Contents and requirements of this thesis were adopted by subject HCM city, 2019 Subject Supervisor ACKNOWLEDGEMENTS The success of the thesis would have been impossible without the significant support from many people Firstly, my wholehearted appreciation goes to my wonderful supervisor I am deeply indebted to Dr Nguyen Huu Hung for giving me a chance to work in the Laboratory of Biochemistry - Biopharmacy - Parasitology and the Laboratory of Stem cells and Cancer cells and his tireless support, invaluable guidance and inspiring supervision through this challenging journey I also wish to express my deep gratitude to all members in laboratory MSc ThiPhuong Nguyen, MSc Thi-Phuong-Thao Le, MSc Thi-Tuyet-Trinh Thai and Ms Ngoc-Kim-Thuy Dang who supported, encouraged, guided me through my study and their advice was invariably wise I am grateful to all friends, especially Thi-Truc-Phuong Nguyen, who have supported me with kindness, intelligence and always be beside me when I need Finally, the heartiest thank to my family for their love, their resilience, their motivation and their belief in me throughout the period of my study Xuan-Thu Chau Faculty of Biotechnology Nguyen Tat Thanh University TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii ABSTRACT iv LIST OF FIGURES V LIST OF TABLES vi LIST OF ACRONYMS vii INTRODUCTION ix CHAPTER LITERATURE REVIEW 1.1 Giant liver fluke Fasciola gigantica 1.1.1 Classification 1.1.2 Morphology 1.1.3 Life cycle 1.1.4 Fascioliasis epidemiology 1.1.5 Symptoms and signs 1.1.6 Diagnosis 1.2 IgG antibodies 1.2.1 Structure of IgG molecule and antigen-antibody interaction 1.2.2 Function of IgG molecule 1.3 Production and purification IgG antibodies 1.3.1 Adjuvant 1.3.2 IgG antibodies purification methods 1.4 Antibody applications 11 1.5 Quantity proteins using Quantity-one analysis software (BioRad) 12 ii 1.6 International and national research 12 1.6.1 International research 12 1.6.2 National research 14 CHAPTER CONTENT ANDMETHODS 15 2.1 Place of administration 15 2.2 Contents 15 2.3 Methods 15 2.3.1 Rabbit immunization 15 2.3.2 Ouchterlony assay 16 2.3.3 Purification of IgG antibodies from immunized rabbit 16 2.3.4 SDS-PAGE 17 2.3.5 Indirect ELISA 18 CHAPTER RESULTS AND DISCUSSION 20 3.1 Detection of specific IgG in rabbit after immunization with F gigantica E/S antigen 20 3.2 Antibodies titration 20 3.3 Immunoprecipitation of rabbit IgG by 45% saturated ammonium sulphate 21 3.4 Affinity chromatography using protein G column for IgG purification 22 3.5 Determination of purified IgG bio-activity byindirect ELISA 23 CONCLUSIONS AND RECOMMENDATIONS 25 REFERENCES 26 APPENDICES 29 iii ABSTRACT Serodiagnosis of fasciolosis for specific IgG detection has a drawback that one can not discriminate a recent infection with that happened years ago or it is difficult to determine if the patients is recovering from infection Hence, the detection of stool parasite antigens, especially excretory/secretory (E/S) antigen is thought to be a better alternative for diagnosis of fasciolosis since it reflects the real parasite burden In this study, rabbit IgG antibodies specific for Fasciola gigantica’s excretory/secretory antigen were produced in rabbit IgG antibodies were then purified by affinity chromatography using protein G column and use tested for it activity by indirect ELISA The results show that 55 mg of IgG was obtained from 60 ml of rabbit serum and had purity of about 95% The purified IgG highly binds to excretory/secretory antigen in ELISA assay indicating for their use for development of a test kit for antigen detection in fasciolosis IV LIST OF FIGURES Figure 1.1 The giant liver fluke Fasciola gigantica morphology Figure 1.2 Fasciola gigantica eggs were observed under an optical microscope Figure 1.3 Life cycle of Fasciola gigantica Figure 1.4 IgG structure Figure 2.1 Diagram summarizing the process of production and purification of rabbit IgG against F gigantica E/S antigens 18 Figure 3.1 The agglutination of F gigantica E/S antigen with antibodies produced in rabbit after immunization 19 Figure 3.2 Titration of F gigantica E/S antigen specific antibodies in rabbit sera after immunization 20 Figure 3.3 SDS-PAGE analysis of IgG after precipitation by 45% saturated ammonium sulphate 21 Figure 3.4 Capture of IgG by affinity chromatography using protein G column 22 Figure 3.5 Determination of purified IgG activity by ELISA The graph shows the OD value 23 V LIST OF TABLES Table 1.1 Summary of some of the terms used to describe antigen - antibody interaction Table 1.2 Affinity of protein A/G for IgG types from human and rabbit 10 Table 2.1 Immunization schedule in rabbit with E/S antigen 16 Table 3.1 IgG antibodies recovered after precipitation with 45% saturated ammonium sulphate 21 vi LIST OF ACRONYMS ADCC: Antibody dependent cellular cytotoxicity APC: Antigen presenting cell APS: Ammonium persulphate AS: Ammonium sulphate BSA: Bovine serum albumin CDC: Center of Disease Control and Prevention dH2O: Distilled water DNA: Deoxyribonucleic acid ELISA: Enzyme-link Immunosorbent Assay EIA: Enzyme Immunosorbent Assay E/S: Excretory/Secretory Fab: Fragment antigen binding Fc: Fragment crystallisable FCA: Freund’s Complete Adjuvant FIA: Freund’s Incomplete Adjuvant HLB: Hydrophile - lipophile balance HRP: Horseradish peroxidase HF: Human Fascioliasis ICC: Immunocytochemistry IEM: Immuno electron microscopy IF: Immunofluorescene IHC: Immunohistochemistry IP: Immunoprecipitation vii ... Thesis’s title: Production and purification of rabbit IgG antibodies against Fasciola gigantica excretory /secretory antigen Objectives - Production of rabbit IgG antibodies against Fasciola gigantica. .. development of parasitic diagnosis products Objectives The study focuses on production and purification of rabbit IgG antibodies against Fasciola gigantica excretory /secretory antigen and assessment of. .. value The thesis ? ?Production and purification of rabbit IgG antibodies against Fasciola gigantica excretory /secretory antigen” was carried out to produce high affinity and specific IgG antibody which