Cho Donghee, Collins Michael T
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA.
Clin Vaccine Immunol. 2006 Oct;13(10):1155-61. doi: 10.1128/CVI.00058-06.
The protein expression profiles and antigenicities of both culture filtrates (CF) and cellular extracts (CE) of Mycobacterium paratuberculosis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), one-dimensional electrophoresis (1-DE) and 2-DE immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The CF proteins were harvested from supernatants of stationary-phase liquid cultures and concentrated by size exclusion filtration. The CE proteins were extracted by mechanical disruption of cells using glass beads and a high-speed agitator. Analysis of SDS-PAGE gels showed that the majority of CF proteins had low molecular masses (<50 kDa), whereas CE protein mass ranged more evenly over a broader range up to 100 kDa. By 2-DE, CF proteins had a narrow array of pI values, with most being between pH 4.0 and 5.5; CE proteins spanned pI values from pH 4.0 to 7.0. The antigenicities of CF and CE proteins were first determined by 1-DE and 2-DE immunoblotting with serum from a cow naturally infected with M. paratuberculosis. The serum reacted strongly to more proteins in the CF than the CE. Sera from 444 infected and 412 uninfected cattle were tested by ELISA with CF and CE as solid-phase antigens. Receiver-operator characteristic curve analysis of the ELISA results showed a significantly greater area under the curve for CF compared to CE (P<0.05). A high degree of variability in protein binding patterns was shown with 1-DE immunoblot analysis with 31 sera from M. paratuberculosis-infected cattle. Collectively, these results indicate that serologic tests for bovine paratuberculosis may be improved by using proteins derived from CF instead of CE. To maximize the diagnostic sensitivity of serologic tests, multiple proteins will be required. Even so, a CF ELISA may not be able to detect all M. paratuberculosis-infected cattle, in particular those in the early stages of infection that have yet to mount an antibody response.
通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、一维电泳（1-DE）和二维免疫印迹以及酶联免疫吸附测定（ELISA），比较了副结核分枝杆菌培养滤液（CF）和细胞提取物（CE）的蛋白质表达谱和抗原性。CF蛋白从稳定期液体培养物的上清液中收获，并通过尺寸排阻过滤进行浓缩。CE蛋白通过使用玻璃珠和高速搅拌器机械破碎细胞来提取。SDS-PAGE凝胶分析表明，大多数CF蛋白具有低分子量（<50 kDa），而CE蛋白的分子量在更宽的范围内（高达100 kDa）分布更为均匀。通过二维电泳，CF蛋白的等电点值范围较窄，大多数在pH 4.0至5.5之间；CE蛋白的等电点值范围从pH 4.0到7.0。首先通过一维电泳和二维免疫印迹，用自然感染副结核分枝杆菌的奶牛血清来确定CF和CE蛋白的抗原性。该血清与CF中的更多蛋白质发生强烈反应，而与CE中的反应较弱。用CF和CE作为固相抗原，通过ELISA对444头感染牛和412头未感染牛的血清进行了检测。ELISA结果的受试者工作特征曲线分析表明，与CE相比，CF的曲线下面积显著更大（P<0.05）。用来自副结核分枝杆菌感染牛的31份血清进行一维免疫印迹分析，结果显示蛋白质结合模式存在高度变异性。总体而言，这些结果表明，使用CF衍生的蛋白质而非CE衍生的蛋白质，可能会改进牛副结核的血清学检测。为了使血清学检测的诊断敏感性最大化，将需要多种蛋白质。即便如此，CF ELISA可能无法检测到所有感染副结核分枝杆菌的牛，特别是那些处于感染早期尚未产生抗体反应的牛。
