Kiguchi Yuki, Oyama Hiroyuki, Morita Izumi, Katayama Emiko, Fujita Masatoshi, Narasaki Mai, Yokoyama Aiko, Kobayashi Norihiro
Kobe Pharmaceutical University.
Biol Pharm Bull. 2018;41(7):1062-1070. doi: 10.1248/bpb.b18-00162.
Antibodies are essential for characterizing various analytes. "Molecular-breeding" approaches enable rapid generation of antibody mutants with desirable antigen-binding abilities. Typically, prototype antibodies are converted to single-chain Fv fragments (scFvs), and random mutations are genetically introduced to construct molecular libraries with a vast diversity. Improved species therein are then isolated via phage display genotype-phenotype-connecting systems to separate them from a large excess of nonspecific scFvs. During these experiments, counting of phage particles is routinely performed. However, current methods depend on the time-consuming overnight cultivation of phage-infected bacteria on agar plates to estimate phage numbers as plaque-forming units (pfu) or colony-forming units, the results of which fluctuate considerably. Immunochemical systems capturing phage particles should be a more convenient and robust alternative. We therefore generated monoclonal antibodies against M13 filamentous phage, which is commonly used for phage display, by employing hybridoma technology. Combinatorial use of two such antibodies (Ab-M13#53 and #71; both specific to the major coat protein pVIII) enabled development of a sandwich enzyme-linked immunosorbent assay (ELISA) that could measure ca. 10-10 phage pfu/mL. To construct a more convenient system, Ab-M13#71 was converted to the scFv form and further fused with an alkaline phosphatase variant. Using this fusion protein, the sandwich ELISA enabled rapid (within 90 min) and reliable phage counting without reducing the sensitivity, and the results were reasonably consistent with those of infection-based methods. The present anti-phage antibodies and scFvs might also enable visualization of individual phage particles by combining them with sensitive fluorescent staining.
抗体对于各种分析物的表征至关重要。“分子育种”方法能够快速产生具有理想抗原结合能力的抗体突变体。通常，将原型抗体转化为单链Fv片段（scFv），并通过基因手段引入随机突变以构建具有广泛多样性的分子文库。然后通过噬菌体展示基因型 - 表型连接系统从大量非特异性scFv中分离出其中改良的种类。在这些实验过程中，常规会对噬菌体颗粒进行计数。然而，目前的方法依赖于在琼脂平板上对噬菌体感染的细菌进行耗时的过夜培养，以将噬菌体数量估计为噬斑形成单位（pfu）或菌落形成单位，其结果波动很大。捕获噬菌体颗粒的免疫化学系统应该是一种更方便且稳健的替代方法。因此，我们通过杂交瘤技术产生了针对常用于噬菌体展示的M13丝状噬菌体的单克隆抗体。两种此类抗体（Ab - M13#53和#71；均对主要衣壳蛋白pVIII具有特异性）的组合使用使得能够开发一种夹心酶联免疫吸附测定（ELISA），该测定能够测量约10 - 10噬菌体pfu/mL。为了构建更方便的系统，将Ab - M13#71转化为scFv形式，并进一步与碱性磷酸酶变体融合。使用这种融合蛋白，夹心ELISA能够在不降低灵敏度的情况下快速（90分钟内）且可靠地进行噬菌体计数，并且结果与基于感染的方法相当一致。目前的抗噬菌体抗体和scFv还可能通过将它们与灵敏的荧光染色相结合，实现对单个噬菌体颗粒的可视化。
