Tay Moon Y F, Lee Chin Chin, Vasudevan Subhash G, Moreland Nicole J
Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School, Singapore, Singapore.
Methods Mol Biol. 2014;1138:161-73. doi: 10.1007/978-1-4939-0348-1_11.
High-affinity antibodies are valuable tools for dengue research. A method for the selection of dengue-specific, human antibody fragments using naïve repertoires displayed on M13 filamentous bacteriophage is described. Naïve repertoires are unbiased, thus enabling the identification of antibodies to dengue structural and nonstructural proteins from the same library. Dengue-specific clones are enriched by binding to an immobilized dengue antigen, followed by washing, elution, and amplification of phage for subsequent rounds of selection. Dengue virus has four antigenically related serotypes, and the serotype of the antigen can be kept constant or alternated during the selection process depending on whether serotype-specific or cross-reactive antibodies are required. After the selection process, clones are screened, and specific clones are identified by phage ELISA and Western blot.
高亲和力抗体是登革热研究的宝贵工具。本文描述了一种利用展示在M13丝状噬菌体上的天然抗体库来选择登革热特异性人抗体片段的方法。天然抗体库没有偏向性，因此能够从同一个文库中鉴定出针对登革热结构蛋白和非结构蛋白的抗体。通过与固定化的登革热抗原结合来富集登革热特异性克隆，随后进行洗涤、洗脱以及噬菌体扩增，以便进行后续轮次的筛选。登革热病毒有四种抗原相关的血清型，在筛选过程中，根据需要的是血清型特异性抗体还是交叉反应性抗体，抗原的血清型可以保持不变或交替变化。筛选过程结束后，对克隆进行筛选，并通过噬菌体ELISA和蛋白质印迹法鉴定特异性克隆。
