Development of a Sandwich ELISA Method for Detecting Mycoplasma Contamination in Cell Culture
- Abstract
- The aim of this study was to develop sandwich ELISA method for the detection of common mycoplasma contaminants in cell culture. We produced the mouse monoclonal antibodies (MAbs) and rabbit antibodies against Mycoplasma arginini, M. hyorhinis, M. orale and Acholeplasma laidlawii. Binding specificities of MAbs and rabbit antibodies to the immunized antigens were confirmed by indirect ELISA and Western blot analysis. Among the established MAbs and rabbit anti-mycoplasmas, MAb MH03 against M. hyorhinis, and rabbit anti-A. laidlawii showed high cross reactivities to the four Mycoplasma species. We constructed a sandwich ELISA using MH03 as capture, horse radish peroxidase labeled rabbit anti-A. laidlawii as detector, and could detect M.arginini with the detection limit of 0.5 ㎍/ml and M. hyorhinis, M. orale and A. laidlawii with the detection limit of 0.1 ㎍/ml. These results indicate that the sandwich ELISA developed in this study could be applied for the detection of the four major Mycoplasma contaminants in cell culture.
- Issued Date
- 2008
- Awarded Date
- 2008. 2
- Type
- Dissertation
- Affiliation
- 제주대학교 대학원
- Department
- 대학원 수의학과
- Advisor
- 임윤규
- Table Of Contents
- Introduction = 1
Materials and Methods = 2
Bacterial strains = 2
Bacterial culture = 2
Antigen preparation = 2
Mouse immunization = 2
Cell fusion for producing hybridoma = 3
Rabbit immunization = 3
Indirect ELISA for the screening of hybridoma and titration of rabbit sera = 3
Purification of MAbs and Rabbit anti-Mycoplasma antibodies = 4
Labeling HRP to Purified antibodies = 4
SDS-Polyacrylamide Gel Electrophresis (SDS PAGE) and Western Blot Analysis(W/B) = 4
Sandwich ELISA for the detection of Mycoplasma = 5
PCR = 5
RESULTS = 6
Preparation of Mycoplasma antigen = 6
Monoclonal antibodies against Mycoplasma species = 7
Rabbit antibodies against Mycoplasma species. = 10
Purification of MAb MH03 = 13
Purification of rabbit specific antibodies = 13
Western blot analysis = 14
Optimization of sandwich ELISA = 15
PCR = 16
Discussion = 18
Reference = 20
- Degree
- Master
- Publisher
- 제주대학교 대학원
- Citation
- Hoa Van Ba. (2008). Development of a Sandwich ELISA Method for Detecting Mycoplasma Contamination in Cell Culture
- Appears in Collections:
- General Graduate School > Veterinary Medicine
- Files in This Item:
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