Wang Na, He Miao, Shi Han-Chang
ESPC State Key Joint Laboratory, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China.
Anal Chim Acta. 2007 May 8;590(2):224-31. doi: 10.1016/j.aca.2007.03.041. Epub 2007 Mar 25.
In order to establish ELISA (enzyme-linked immunosorbent assay) method to detect Total E. coli in water environment, E. coli multi-characters antigens in water environment were prepared according to the characters of kinds of E. coli serotypes, including antigen of whole cell, antigen of disrupted whole cell, somatic antigen, flagellar antigen and fimbrial antigen. Total E. coli polyclonal antibodies were obtained from the New Zealand rabbits immunized with these five antigens, respectively. Antibodies generated in this research are with high titers and good purity, can conjugate with antigens, specifically, stably and strongly. Indirect ELISA shows the titers of antibody of whole cell and antibody of disrupted whole cell are both over 1x10(5). The cross-reactivity of the antibody is from 12 to 30% which indicate the specificity of the antibody against Total E. coli. Based on these antibodies, we established indirect ELISA method to detect Total E. coli in water environment. The matrix effects were studied and the results show that there is no significant influence by all the factors. The ELISA result shows that the detection limitation could be 10(4) CFU (colony forming units) L(-1). The indirect ELISA method developed in this study is well suited for Total E. coli analysis in real water samples as a rapid screen method.
为建立检测水环境中总大肠杆菌的酶联免疫吸附测定（ELISA）方法，根据多种大肠杆菌血清型的特性制备了水环境中大肠杆菌多特征抗原，包括全细胞抗原、破碎全细胞抗原、菌体抗原、鞭毛抗原和菌毛抗原。分别用这五种抗原免疫新西兰兔，获得了总大肠杆菌多克隆抗体。本研究产生的抗体具有高滴度和良好的纯度，能与抗原特异性、稳定且强烈地结合。间接ELISA结果显示全细胞抗体和破碎全细胞抗体的滴度均超过1×10⁵。抗体的交叉反应率为12%至30%，表明该抗体对总大肠杆菌具有特异性。基于这些抗体，建立了检测水环境中总大肠杆菌的间接ELISA方法。研究了基质效应，结果表明所有因素均无显著影响。ELISA结果显示检测限可达10⁴CFU（菌落形成单位）L⁻¹。本研究建立的间接ELISA方法作为一种快速筛选方法，非常适合实际水样中总大肠杆菌的分析。
