Rodgers S J, Saliki J T, Blouin E F, Kocan K M
Oklahoma Animal Disease Diagnostic Laboratory, Oklahoma State University, Stillwater 74076-7001, USA.
Ann N Y Acad Sci. 1998 Jun 29;849:282-92. doi: 10.1111/j.1749-6632.1998.tb11060.x.
Serologic diagnosis of anaplasmosis is currently done by the complement-fixation, ELISA, and card agglutination tests. These tests have utilized A. marginale harvested from bovine erythrocytes as antigen which is often contaminated with erythrocyte stroma. We are currently testing A. marginale propagated in a Ixodes scapularis cell line as antigen for serologic tests. In this study, we report the use of the cell culture-derived A. marginale as antigen for development of a rapid, semi-automated latex agglutination test. Diluted serum and latex (polystyrene microspheres), sensitized with cell culture-derived A. marginale proteins, were dispensed into 96-well microtiter plates. An initial reading of light transmission was recorded by a computer-interfaced scanning autoreader. After 30 minutes, the plates were mixed and read a second time, recording the delta % light transmittance. The sensitized latex microspheres (latex) agglutinated in the presence of A. marginale antibodies, thus producing an increase in light transmittance. In preliminary tests, 724/977 of the sera were positive for A. marginale antibodies with an apparent agreement of 83.3% when compared with the complement-fixation test. Sensitization and sera dilution buffers were shown to have a marked effect on the sensitivity and specificity of this assay. Results will be presented on the optimization of buffers and the testing of sera from experimentally and field-infected cattle.
无形体病的血清学诊断目前通过补体结合试验、酶联免疫吸附测定和卡片凝集试验进行。这些试验使用从牛红细胞中收获的边缘无形体作为抗原，而该抗原常被红细胞基质污染。我们目前正在测试在肩突硬蜱细胞系中繁殖的边缘无形体作为血清学检测的抗原。在本研究中，我们报告了使用细胞培养来源的边缘无形体作为抗原开发一种快速、半自动乳胶凝集试验。将用细胞培养来源的边缘无形体蛋白致敏的稀释血清和乳胶（聚苯乙烯微球）加入96孔微量滴定板中。通过计算机接口扫描自动读数仪记录初始透光率读数。30分钟后，将平板混合并再次读数，记录透光率变化百分比。致敏乳胶微球（乳胶）在存在边缘无形体抗体时发生凝集，从而导致透光率增加。在初步试验中，与补体结合试验相比，977份血清中有724份边缘无形体抗体呈阳性，表观符合率为83.3%。已证明致敏和血清稀释缓冲液对该检测的敏感性和特异性有显著影响。将展示关于缓冲液优化以及来自实验感染和现场感染牛的血清检测的结果。
