톡소포자충의 lactate dehydrogenase를 이용한 단클론 항체의 생산
Production of Monoclonal Antibodies for Toxoplasma gondii Lactate Dehydrogenase
- 주제(키워드) Toxoplasma gondii , lactate dehydrogenase , cell fusion technique , monoclonal antibodies
- 발행기관 아주대학교
- 지도교수 신호준
- 발행년도 2009
- 학위수여년월 2009. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000009588
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Background/Purpose: Toxoplasma gondii is one of the most common parasitic infections of human and other warm blooded animals. Because of the geographical distribution of toxoplasmosis, there is a need to develop a sensitive, rapid, simple and an inexpensive test for the early screening of patiens. In this study, to create the dipstick kit that has high sensitivity against LDH (lactate dehydrogenase) of T. gondii, at the first of all, hybridoma cell lines producing monoclonal antibodies with high affinity and specificity against T. gondii LDH (TgLDH) were established. Methods: After 6-8 week old BALB/c mice were immunized with synthetic peptides of TgLDH, splenocytes were harvested. To get the monoclonal antibodies (McAbs), splenocytes were fused with myeloma cells using PEG by a cell fusion technique. Hybridoma cells were selected by ELISA and subcloned through a limiting dilution method. McAbs were characterized by isotyping. Results: Eight McAb-producing hybridoma cells were selected. After three times of limiting dilution, eight clones, 1A10, 1B12, 3B2, 3B6, 1C12, 3C8, 3G6 and 3H1, were selected. OD values of eight monoclonal antibodies were ranged from 0.938 to 1.269 by ELISA. All eight McAbs isotypes were IgM class. Conclusion: These results indicated that eight hybridoma clones had strong reactivity to TgLDH and may be useful for diagnosis to T. gondii infection.
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TABLE OF CONTENTS
ABSTRACT................i
TABLE OF CONTENTS.................ii
LIST OF FIGURES.......v
LIST OF TABLE......... vi
I. INTRODUCTION...... 1
A. Morphology........... 1
B. Life cycle.............. 4
C. Diagnosis............. 7
D. Lactate dehydrogenase of parasites......... 7
E. Purpose of this study............. 8
II. MATERIALS AND METHODS... 9
A. Culture of myeloma cells........ 9
B. Antigen..9
C. Immunization of mice with TgLDH........... 9
D. Cell fusion.............10
E. ELISA screening.... 11
F. Selection of hybridoma cells....11
G. Large scale production of McAbs............ 12
H. Monoclonal antibody isotyping.12
III. RESULTS..............13
A. Antibody formation of immunized mice 13
B. Cell fusion.............15
C. Isolation of antibody-producing hybridoma cells........ 16
D. Producing large scale of antibodies..........17
E. The specificity of McAbs with T. gondii lysate...........18
F. Isotyping of monoclonal antibodies............20
IV. DISCUSSION........ 21
V. CONCLUSION........23
REFERENCES........... 24
국문요약....29
LIST OF FIGURES
Fig. 1. Morphology of T. gondii.... 3
Fig. 2. Life cycle of T. gondii ...... 6
Fig. 3. Polyclonal antibody titers from the TgLDH-5-immunized mice........ 14
Fig. 4. Antibody titers of the the hybridoma cell culture supernatant after cell fusion ........15
Fig. 5. Antibody titers of the hybridoma cell culture supernatant after third limiting dilution .......... 16
Fig. 6. Antibody titers of McAb-containing ascitic fluids.. 17
Fig. 7. Antibody titers of the hybridoma cell culture supernatant using T. gondii lysate as antigen................18
Fig. 8. Antibody titers of McAb-containing ascitic fluids using T. gondii lysate as antigen..19
LIST OF TABLE
Table 1. Amino acids sequence of synthetic peptide for TgLDH-5... 9
Table 2. Isotype profile of eight hybridoma clones producing monoclonal antibody...20
