豚鼠气单胞菌胶体金免疫层析试纸条的研制  

A rapid immunochromatography test format for Aeromonas caviae detection

作　　者：辛志明[1] 樊海平[1] 吴斌[1] 张新艳[1] 

机构地区：[1]福建省淡水水产研究所,福建福州350002

出　　处：《水产学报》2009年第4期679-684,共6页Journal of Fisheries of China

基　　金：海洋与渔业科技项目(闽海渔科05201号)

摘　　要：采用淋巴细胞杂交瘤技术制备抗豚鼠气单胞菌单克隆抗体分泌细胞株,获取抗豚鼠气单胞菌单克隆抗体,柠檬酸三钠还原氯金酸制备胶体金颗粒,选择直径20 nm的胶体金颗粒,标记抗豚鼠气单胞菌单克隆抗体并制备胶体金垫。将胶体金垫与喷涂有抗豚鼠气单胞菌兔多克隆抗体和羊抗鼠抗体的硝酸纤维素膜及样品吸收垫等组装成免疫层析试纸条,建立豚鼠气单胞菌的快速检测方法。用灭活细菌与血清混合的模拟样本测定试纸条的特异性、灵敏度,结果显示,试纸条对嗜水气单胞菌、温和气单胞菌、鳗弧菌、溶藻弧菌、荧光假单胞菌、副溶血弧菌等13种常见病原菌没有交叉反应,与豚鼠气单胞菌显示特异性反应,检测灵敏度为1×106CFU/mL,结果显示时间小于5 min。研制的豚鼠气单胞菌胶体金免疫层析检测试纸条具有快速、简便、特异性高、适用于基层临床生产推广应用等优点。Aeromonas caviae is a pathogenic bacterium that can infect various farmed fishes, and cause high mortality. Thus, a rapid and sensitive technique is necessary for the detection of the bacteria to prevent further economic losses. For rapid and simple examination, an immunochromatographic lateral flow assay system （GICA） was developed. In GICA, the rabbit anti-A, caviae antibody applied to nitrocellose membrane acts as a capture reagent for the target analyte in a sample, and the monoclonai antibody against A. caviae conjugated with colloidal gold acts as signal generator. Sodium citrate was used as reducer to donate electrons to the positively charged gold ions in solution and colloidal gold particles was produced. The size of colloidal gold particles was checked by a transmission electron microscope （ TEM）, and the TEM images showed the average diameter of colloidal gold particles was almost the same size： approximately 20.0 nm in diameter. We produced monoclonal antibody against A. cavia by hybridoma technology. After cloning, three strains of hybridoma named 0El0, 1C4 and 1F10 were obtained. An absorbing pad, often paper, is attached to notrocellose membrane. On the nitrocellose membrane, rabbit anfi-A, caviae antibody was used as a capture antibody at the test line （T） and goat anti-mouse IgG antibody was used as the capture antibody at the control line （C）. A conjugate pad which was attached to the notrocellose membrane contains gold particles conjugated with 1F10 monoclonal antibody specific to the analyte being detected. A sample pad, usually glass fiber, is attached to the conjugate pad. During detection, the liquid sample migrates by capillary diffusion through the conjugate pad, rehydrating the gold conjugate and allowing the interaction of the sample analyte with the conjugate. The complex of gold conjugate and analyte then moves onto the notrocellose membrane and migrates towards the test line （T）, where it becomes immobilized and produces a distinct signal in the fo

关 键 词：豚鼠气单胞菌 杂交瘤技术 单克隆抗体 胶体金 免疫层析 

分 类 号：S917[农业科学—水产科学]