- 學位論文
沙門氏菌之單株抗體的生產與分析研究
Production and Characterization of Monoclonal Antibodies Directed to Salmonella enterica
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摘要
沙門氏菌（Salmonella enterica）是造成全球食物中毒的最主要病原菌之一，不但威脅人類的健康，也會造成社會問題及經濟損失，因此發展快速、準確的沙門氏菌檢驗方法有其必要性。目前我國對沙門氏菌的標準檢驗方法是使用傳統的細菌培養法，缺點是完成檢驗所需的時間較長，且耗費較多的人力與空間。故本研究擬生產可辨識沙門氏菌的專一抗體，以利後續更新沙門氏菌的檢測方法，或是做為沙門氏菌的基礎研究工具之用。 在本研究中，我們以沙門氏菌（Typhimurium血清型）的細胞壁溶出物或以超音波震盪處理的破菌樣品做為抗原，分別對小鼠進行免疫注射，經過三次細胞融合及選殖實驗後，總共篩選得到七株可辨識沙門氏菌的單株抗體：其中的一株（I-1）除了可辨識沙門氏菌之外，也會辨識各種測試的革蘭氏陽性菌及革蘭氏陰性菌；一株（II-1）除了可辨識沙門氏菌之外，也會辨識腸菌科中的大腸桿菌及志賀氏桿菌；兩株（III-1、III-2）會對沙門氏菌的Typhimurium和Enteritidis兩種血清型產生反應，但不會對其他腸菌科的細菌作用；另三株（IV-1、IV-2、IV-3）則只會辨識沙門氏菌中的Typhimurium血清型菌株。這七株抗體中，有四株（III-2、IV-1、IV-2、IV-3）的辨識抗原，經檢測後確定為lipopolysaccharide (LPS)上的O antigen：其中一株（III-2）專一辨識沙門氏菌的O12 antigen；其餘三株則是辨識O5 antigen。 本研究所生產的七種單株抗體，依其專一性及結合力的差別，或可做為基礎研究的工具，如不同菌種的親緣關係探討或特定抗原的鑑定；亦可被應用於病原菌檢測方法的開發或功能性食品的生產。此外，我們也開發了一種新的免疫檢測方式，藉簡易的離心濃縮步驟，將細菌的偵測極限降低至104 CFU/mL，偵測時間僅需2.5 h以內。未來透過偵測敏感度的進一步提升，應有取代當前國家標準方法的潛力。
並列摘要
Salmonella enterica is one of the main pathogens causing foodborne disease in the world. It not only threaten people’s health, but also result in social problems and economic losses. Therefore it is important to develop a rapid and accurate detection method for Salmonella. Currently, the national standard detection methods in Taiwan employ the traditional bacterial culture method, which is time consuming, labor extensive, and space required. Thus, the aim of the present study is to produce Salmonella-specific monoclonal antibodies, which may be used as tools for basic researches and for development of immunochemical detection method for Salmonella. To fulfille our aim, we used detergent-dissolved cell wall components or sonicated bacterial cells (Salmonella enterica, serovar Typhimurium) as antigens to immunize mice. After three independent cell fusion and cloning processes, we have successfully selected seven Salmonella-detecting monoclonal antibodies (MAbs). Among them, one (I-1) recognizes not only Salmonella but also all the other tested Gram-positive and Gram-negative bacteria; one (II-1) can detect Salmonella and two other enteric bacteria (Escherichia coli and Shigella sonnei); two (III-1 and III-2) can react with Salmonella enterica of two different serotypes (Typhimurium and Enteritidis), but not other bacteria in Enterobacteriaceae; while the last three (IV-1, IV-2 and IV-3) can only recognize the Typhimurium serotype of Salmonella enterica (Typhimurium-specific). Results of antigen scrutinization revealed that four of generated MAbs (III-2, IV-1, IV-2 and IV-3) detect recognize O antigens: MAb III-2 reacts with Salmonella O12 antigen, while the other three detect the O5 antigen. Based upon the difference in their specificity and antigen-binding capability, these seven MAbs might be applicable for basic researches, e.g. bacterial phylogenetic, structure and function, and pathogenesis studies. In addition, they may be used to develop bacterial detection methods or be used as supplement of functional foods. Presently, we have developed a microfuge tube-based immunoassay which can detect Salmonella at 104 CFU/mL within 2.5 h. By increasing the sensitivity of this assay, it has the potential to develop a new immunochemical detection method for Salmonella.
參考文獻
Amaro M, Oaew S, Surareungchai W. 2012. Scano-magneto immunoassay based on carbon nanotubes/gold nanoparticles nanocomposite for Salmonella enterica serovar Typhimurium detection. Biosensors & bioelectronics
Anzai Y, Kim H, Park JY, Wakabayashi H, Oyaizu H. 2000. Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. International journal of systematic and evolutionary microbiology 50 Pt 4: 1563-89
Blais BW, Martinez-Perez A. 2008. Detection of group D salmonellae including Salmonella Enteritidis in eggs by polymyxin-based enzyme-linked immunosorbent assay. Journal of food protection 71: 392-6
Bravo D, Hoare A, Silipo A, Valenzuela C, Salinas C, et al. 2011. Different sugar residues of the lipopolysaccharide outer core are required for early interactions of Salmonella enterica serovars Typhi and Typhimurium with epithelial cells. Microbial pathogenesis 50: 70-80
Busse M. 1995. Media for Salmonella. International journal of food microbiology 26: 117-31
延伸閱讀
- 江宗明、陳明珠、王貴譽（1983）。沙門氏桿菌病之臨床觀察與分析。Acta Paediatrica Sinica，24(3)，159-163。https://www.airitilibrary.com/Article/Detail?DocID=00016578-198309-24-3-159-163-a
- 蔡美英、張瑞烽、劉耀輝、彭健芳（1985）。沙門氏桿菌中抗藥性的探討。The Kaohsiung Journal of Medical Sciences，1(5)，310-320。https://doi.org/10.6452/KJMS.198505.0310
- 曾少鋒（2013）。台灣環境水體沙門氏桿菌分離鑑定暨抗 藥性及整合子分析〔碩士論文，國立中正大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0033-2110201613535234
- Ng, S. P. (1999). A new approach to Salmonella detection and serogroup differentiation using murine monoclonal antibodies [doctoral dissertation, The University of Hong Kong]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0029-1812201200004587
- Luk, M. C. (1987). The production and characterization of murine monoclonal antibodies against Salmonella lipopolysaccharide [master's thesis, The University of Hong Kong]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0029-1812201200005589