| Summary: | 碩士 === 國立臺灣師範大學 === 生命科學研究所 === 100 === Escherichia coli is one of the main pathogens causing human disease. It annually not only made people’s deadth, but also result in large economic loss in the world.Therefore it is important to develop a rapid and accurate detection method for E. coli. Currently, the national standard detection method of E. coli is bacterial culture method, which is time-consuming, labor extensive, and space required. Thus, the aim of the present study is to produce E. coli-specific monoclonal antibodies, which may be used to improve the detection method for E. coli and as tools for basic researches.
To fulfille our aim, we used sonicated E. coli(ATCC 11775, O1：K1：H7) cells as antigens to immuniz mice and manufactured monoclonal antibodies (MAbs) against E. coli. After cell fusion and cloning processes, we have successfully obtained nine E. coli-detecting MAbs. We can cluster these MAbs into two groups by bacterial specificity. The group I MAbs (I-1 ~ I-6) recognize just E. coli, and the group II MAbs (II-1 ~ II-3) recognize not only E. coli, but also all the other tested Gram-positive and Gram-negative bacteria.
The group I MAbs recognize the O-antigen of LPS on the surface of E. coli. The result of immunoprecipitation proved the group II MAbs recognize antigens are cytosolic or or secreted out by bacteria. The group I MAbs might be used to develop E. coli immunodetection methods or as funtional antibiotic supplyment of food produced by genetic engineering. Besides, they can be used as a tool for bacterial O-antigen analysis.
Then, the group II MAbs might be applicable for detection of total bacterial count or basic researches, e.g. bacterial phylogenetic studies.
In this study, we presently used our I-5 MAb to develop a pilot study of immunodetection method. In this microfuge tube-based immunoassay, we used simple processes to increase the sensitivity at 104 CFU/mL within 2.5 h. After characterizing the specificity of MAbs and increasing the sensitivity of this assay in the future, this method might have the potential to replace the national detection method for E.coli.
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