Grant I R, Ball H J, Rowe M T
Department of Food Science (Food Microbiology), The Queen's University of Belfast, Belfast BT9 5PX, United Kingdom.
Appl Environ Microbiol. 1998 Sep;64(9):3153-8. doi: 10.1128/AEM.64.9.3153-3158.1998.
An immunomagnetic separation (IMS) technique was developed to facilitate selective isolation of Mycobacterium paratuberculosis cells from milk. Rabbit polyclonal antibodies against radiation-killed intact M. paratuberculosis cells were produced and used to coat sheep anti-rabbit immunoglobulin G (IgG) type M-280 Dynabeads. The rabbit anti-M. paratuberculosis IgG-coated beads (IMB) reacted strongly with laboratory strains of M. paratuberculosis as determined by slide agglutination, and microscopic examination confirmed that M. paratuberculosis cells attached to the IMB. The IMB were found to have a maximum binding capacity of 10(4) to 10(5) CFU of M. paratuberculosis. Studies showed that IMS selectively recovered M. paratuberculosis from inoculated milk containing as few as 10 CFU of M. paratuberculosis per ml when 10 microliter IMB (ca. 10(6) beads) was added to 1 ml of milk and the preparation was incubated for 30 min at room temperature with gentle agitation. Larger volumes of milk (10 and 50 ml) were centrifuged and resuspended in 1 ml of phosphate-buffered saline-0.05% Tween 20 prior to IMS in order to increase the sensitivity of the method. Currently, primary isolation of M. paratuberculosis from a milk sample relies on chemical decontamination, followed by culturing on Herrold's egg yolk medium, which must be incubated at 37 degreesC for up to 18 weeks. The potential value of our IMS method is as an aid for rapid detection of M. paratuberculosis in milk when it is used in conjunction with end point detection methods, such as IS900 PCR or an enzyme-linked immunosorbent assay.
开发了一种免疫磁分离（IMS）技术，以促进从牛奶中选择性分离副结核分枝杆菌细胞。制备了针对经辐射杀死的完整副结核分枝杆菌细胞的兔多克隆抗体，并用于包被羊抗兔免疫球蛋白G（IgG）型M-280 Dynabeads磁珠。通过玻片凝集测定，兔抗副结核分枝杆菌IgG包被的磁珠（IMB）与副结核分枝杆菌的实验室菌株强烈反应，显微镜检查证实副结核分枝杆菌细胞附着在IMB上。发现IMB对副结核分枝杆菌的最大结合能力为10⁴至10⁵CFU。研究表明，当向1 ml牛奶中加入10 μl IMB（约10⁶个磁珠）并在室温下轻轻搅拌孵育30分钟时，IMS能从每毫升含低至10 CFU副结核分枝杆菌的接种牛奶中选择性回收副结核分枝杆菌。为了提高该方法的灵敏度，在进行IMS之前，将较大体积的牛奶（10 ml和50 ml）离心并重悬于1 ml含0.05%吐温20的磷酸盐缓冲盐水中。目前，从牛奶样品中初次分离副结核分枝杆菌依赖于化学去污，然后在赫罗尔德蛋黄培养基上培养，该培养基必须在37℃下孵育长达18周。当我们的IMS方法与终点检测方法（如IS900 PCR或酶联免疫吸附测定）结合使用时，其潜在价值在于有助于快速检测牛奶中的副结核分枝杆菌。
