Yakes Betsy Jean, Lipert Robert J, Bannantine John P, Porter Marc D
Department of Chemistry, Ames Laboratory-USDOE, and Institute for Combinatorial Discovery, Iowa State University, Ames, Iowa 50011, USA.
Clin Vaccine Immunol. 2008 Feb;15(2):227-34. doi: 10.1128/CVI.00334-07. Epub 2007 Dec 12.
A sandwich immunoassay for the rapid, low-level detection of Mycobacterium avium subsp. paratuberculosis has been developed. M. avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle, and one of the major obstacles in controlling the spread of this disease is the inability to rapidly detect small amounts of bacteria or other diagnostic markers shed during the subclinical stage of infection. This paper details the development and performance of an assay for sonicated M. avium subsp. paratuberculosis lysate that is based on surface-enhanced Raman scattering (SERS). There are two key components of the assay: (i) an immobilized layer of monoclonal antibodies that target a surface protein on the microorganism; and (ii) extrinsic Raman labels (ERLs) that are designed to selectively bind to captured proteins and produce large SERS signals. By correlating the number of M. avium subsp. paratuberculosis bacilli present prior to sonication to the amount of total protein in the resulting sonicate, the detection limit determined for total protein can be translated to the microorganism concentration. These findings yield detection limits of 100 and 200 ng/ml (estimated to be 500 and 1,000 M. avium subsp. paratuberculosis bacilli/ml) for sonicate spiked in phosphate buffer and sonicate spiked in whole milk, respectively. Moreover, the time required to complete the assay, which includes sample preparation, antigen extraction, ERL incubation, and readout, is less than 24 h. The potential for incorporation of this novel assay into diagnostic laboratories is also briefly discussed.
已开发出一种用于快速、低水平检测副结核分枝杆菌的夹心免疫测定法。副结核分枝杆菌是牛约翰氏病的病原体，控制这种疾病传播的主要障碍之一是无法快速检测出在感染亚临床阶段排出的少量细菌或其他诊断标志物。本文详细介绍了一种基于表面增强拉曼散射（SERS）的副结核分枝杆菌超声裂解物测定法的开发和性能。该测定法有两个关键组成部分：（i）靶向微生物表面蛋白的单克隆抗体固定层；（ii）设计用于选择性结合捕获蛋白并产生大SERS信号的外在拉曼标记（ERL）。通过将超声处理前存在的副结核分枝杆菌杆菌数量与所得超声裂解物中的总蛋白量相关联，可将总蛋白的检测限转化为微生物浓度。这些结果得出，在磷酸盐缓冲液中加标的超声裂解物和在全脂牛奶中加标的超声裂解物的检测限分别为100和200 ng/ml（估计为500和1000副结核分枝杆菌杆菌/ml）。此外，完成该测定所需的时间，包括样品制备、抗原提取、ERL孵育和读数，不到24小时。还简要讨论了将这种新型测定法纳入诊断实验室的可能性。
