Karoonuthaisiri Nitsara, Charlermroj Ratthaphol, Uawisetwathana Umaporn, Luxananil Plearnpis, Kirtikara Kanyawim, Gajanandana Oraprapai
National Center for Genetic Engineering and Biotechnology, BIOTEC, National Science and Technology Development Agency, Pathumthani, Thailand.
Biosens Bioelectron. 2009 Feb 15;24(6):1641-8. doi: 10.1016/j.bios.2008.08.026. Epub 2008 Aug 26.
Pathogenic bacterial contaminations present serious problems for food industry and public health. Rapid, accurate and affordable assays are needed. In this study, antibody arrays to simultaneously detect two foodborne pathogenic bacteria (Escherichia coli O157:H7 and Salmonella spp.) have been developed using chemiluminescent detecting system. Solid supports using nitrocellulose membrane and poly-l-lysine (PLL) glass slide were compared and optimized for antibody array construction. Many parameters including optimal concentrations of antibodies, blocking reagents, assay time, storage time, sensitivity and cross-reactivity were considered during optimization. This study revealed that the PLL slide was a more suitable support due to highly accurate results and the absence of non-specific background. Phosphate-buffered saline (PBS, pH 7.2) and 3% skim milk in PBS buffer were optimal spotting and blocking reagents, respectively. With the same sensitivity for bacterial detection as in a conventional ELISA (10(5)-10(6)CFU/ml for the E. coli O157:H7 and 10(6)-10(7)CFU/ml for Salmonella detections), this antibody array has advantages of a much shorter assay time of 1h and much lower required amounts of antibodies. Moreover, there was no cross-reactivity in the detection among bacteria tested in this study. Bacteria detection in food sample was feasible as demonstrated using bacteria-added milk.
致病性细菌污染给食品工业和公众健康带来了严重问题。因此需要快速、准确且经济实惠的检测方法。在本研究中，利用化学发光检测系统开发了用于同时检测两种食源性病原体（大肠杆菌O157:H7和沙门氏菌属）的抗体芯片。比较并优化了使用硝酸纤维素膜和聚-L-赖氨酸（PLL）玻片的固体支持物用于构建抗体芯片。在优化过程中考虑了许多参数，包括抗体的最佳浓度、封闭剂、检测时间、储存时间、灵敏度和交叉反应性。本研究表明，PLL玻片是更合适的支持物，因为其结果高度准确且不存在非特异性背景。磷酸盐缓冲盐水（PBS，pH 7.2）和PBS缓冲液中的3%脱脂牛奶分别是最佳的点样和封闭试剂。该抗体芯片对细菌检测的灵敏度与传统ELISA相同（检测大肠杆菌O157:H7时为10⁵ - 10⁶CFU/ml，检测沙门氏菌时为10⁶ - 10⁷CFU/ml），具有检测时间短至1小时且所需抗体量少得多的优点。此外，本研究中测试的细菌之间在检测时没有交叉反应。如添加细菌的牛奶所示，在食品样品中检测细菌是可行的。
