大腸桿菌是造成人類感染疾病的重要病原菌之一，每年都造成全球許多生命及經濟的損失，故開發快速且準確的大腸桿菌檢測方法有其必要性。現今我國使用傳統的細菌培養法作為大腸桿菌檢測的標準檢驗方法，其缺點是過程需時冗長，且需使用較多空間與耗費較多人力。故本研究擬生產可專一辨識大腸桿菌的單株抗體，期能用以開發大腸桿菌的免疫檢測方法，或是作為大腸桿菌的基礎研究工具。
本研究以超音波破菌處理過的大腸桿菌(ATCC 11775，血清型O1：K1：H7)為抗原，對小鼠進行免疫注射，經細胞融合及選殖過程後，共獲得9株單株抗體。依照其對不同菌種的作用特性，這9株抗體可被分類為兩群：第一群共有6株 (I-1 ~ I-6)，僅會與大腸桿菌反應；第二群則有3株 (II-1 ~ II-3)，除了對大腸桿菌之外，亦會與本研究所測試的其他10種革蘭氏陰、陽性菌作用。
進一步的抗原成分分析結果顯示，第一群抗體所辨識的抗原應為大腸桿菌表面的脂多醣O抗原；第二群抗體則可能辨識細菌的細胞內或分泌性的成分。在應用上，第一群單株抗體具有開發大腸桿菌免疫檢測方法的潛力，亦可透過遺傳工程技術產生重組抗體後，用作為抑菌用途的功能性食品添加成分，此外，本群抗體還可被用作為分析細菌表面O抗原種類之工具；第二群單株抗體則可應用於生菌數的檢測或被用來分離細菌的保守成分，以供親緣關係探討之用。
本研究也利用自行生產的單株抗體(I-5)，進行大腸桿菌免疫偵測方法的先驅研究。藉著簡易的步驟，我們開發的微量離心管免疫偵測方法可將大腸桿菌的偵測極限下降至104 CFU/mL，並且整個過程僅需2.5小時即可完成，未來若能進一步確定抗體的專一性及提升檢測方法的敏感度，則將有取代現行標準檢測方法的潛力。

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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