School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, Belfast, Northern Ireland, United Kingdom.
J Clin Microbiol. 2012 May;50(5):1598-605. doi: 10.1128/JCM.05747-11. Epub 2012 Feb 8.
This study describes the development and optimization of an immunomagnetic separation (IMS) method to isolate Mycobacterium bovis cells from lymph node tissues. Gamma-irradiated whole M. bovis AF2122/97 cells and ethanol-extracted surface antigens of such cells were used to produce M. bovis-specific polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate M. bovis-specific peptide ligands by phage display biopanning. The various antibodies and peptide ligands obtained were used to coat MyOne tosyl-activated Dynabeads (Life Technologies), singly or in combination, and evaluated for IMS. Initially, M. bovis capture from Middlebrook 7H9 broth suspensions (concentration range, 10 to 10(5) CFU/ml) was evaluated by IMS combined with an M. bovis-specific touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for M. bovis in broth were those coated simultaneously with a monoclonal antibody and a biotinylated 12-mer peptide. These dually coated beads exhibited minimal capture (mean of 0.36% recovery) of 12 other Mycobacterium spp. occasionally encountered in veterinary tuberculosis (TB) diagnostic laboratories. When the optimized IMS method was applied to various M. bovis-spiked lymph node matrices, it demonstrated excellent detection sensitivities (50% limits of detection of 3.16 and 57.7 CFU/ml of lymph node tissue homogenate for IMS-PCR and IMS-culture, respectively). The optimized IMS method therefore has the potential to improve isolation of M. bovis from lymph nodes and hence the diagnosis of bovine tuberculosis.
本研究描述了一种免疫磁分离（IMS）方法的开发和优化，用于从淋巴结组织中分离牛分枝杆菌细胞。γ辐照的全牛分枝杆菌 AF2122/97 细胞和此类细胞的乙醇提取表面抗原被用于在兔子和小鼠中产生牛分枝杆菌特异性多克隆和单克隆抗体。它们还被用于通过噬菌体展示生物淘选产生牛分枝杆菌特异性肽配体。获得的各种抗体和肽配体被用于单独或组合涂覆 MyOne tosyl 活化 Dynabeads（Life Technologies），并进行 IMS 评估。最初，通过 IMS 结合牛分枝杆菌特异性降落 PCR 评估了从 Middlebrook 7H9 肉汤悬浮液（浓度范围为 10 至 10（5）CFU/ml）中捕获牛分枝杆菌的情况。IMS-PCR 结果，随后是 IMS 培养结果表明，在肉汤中对牛分枝杆菌具有最强免疫捕获能力的珠子是同时涂覆单克隆抗体和生物素化 12 肽的珠子。这些双重涂覆的珠子对兽医结核病（TB）诊断实验室中偶尔遇到的 12 种其他分枝杆菌的捕获量最小（平均回收量为 0.36%）。当优化的 IMS 方法应用于各种牛分枝杆菌污染的淋巴结基质时，它表现出优异的检测灵敏度（分别为 IMS-PCR 和 IMS 培养的淋巴结组织匀浆的 50%检测限为 3.16 和 57.7 CFU/ml）。因此，优化的 IMS 方法有可能改善从淋巴结中分离牛分枝杆菌，从而提高牛结核病的诊断。
