Jiang Wenzhi, Cossey Sarah, Rosenberg Julian N, Oyler George A, Olson Bradley J S C, Weeks Donald P
BMC Plant Biol. 2014 Sep 25;14:244. doi: 10.1186/s12870-014-0244-0.
Cell walls are essential for most bacteria, archaea, fungi, algae and land plants to provide shape, structural integrity and protection from numerous biotic and abiotic environmental factors. In the case of eukaryotic algae, relatively little is known of the composition, structure or mechanisms of assembly of cell walls in individual species or between species and how these differences enable algae to inhabit a great diversity of environments. In this paper we describe the use of camelid antibody fragments (VHHs) and a streamlined ELISA assay as powerful new tools for obtaining mono-specific reagents for detecting individual algal cell wall components and for isolating algae that share a particular cell surface component.
To develop new microalgal bioprospecting tools to aid in the search of environmental samples for algae that share similar cell wall and cell surface components, we have produced single-chain camelid antibodies raised against cell surface components of the single-cell alga, Chlamydomonas reinhardtii. We have cloned the variable-region domains (VHHs) from the camelid heavy-chain-only antibodies and overproduced tagged versions of these monoclonal-like antibodies in E. coli. Using these VHHs, we have developed an accurate, facile, low cost ELISA that uses live cells as a source of antigens in their native conformation and that requires less than 90 minutes to perform. This ELISA technique was demonstrated to be as accurate as standard ELISAs that employ proteins from cell lysates and that generally require >24 hours to complete. Among the cloned VHHs, VHH B11, exhibited the highest affinity (EC50 < 1 nM) for the C. reinhardtii cell surface. The live-cell ELISA procedure was employed to detect algae sharing cell surface components with C. reinhardtii in water samples from natural environments. In addition, mCherry-tagged VHH B11 was used along with fluorescence activated cell sorting (FACS) to select individual axenic isolates of presumed wild relatives of C. reinhardtii and other Chlorphyceae from the same environmental samples.
Camelid antibody VHH domains provide a highly specific tool for detection of individual cell wall components of algae and for allowing the selection of algae that share a particular cell surface molecule from diverse ecosystems.
细胞壁对于大多数细菌、古菌、真菌、藻类和陆地植物至关重要，它能赋予细胞形状、维持结构完整性，并保护细胞免受众多生物和非生物环境因素的影响。就真核藻类而言，对于单个物种或不同物种间细胞壁的组成、结构或组装机制，以及这些差异如何使藻类能够栖息于多种多样的环境，我们所知甚少。在本文中，我们描述了使用骆驼科动物抗体片段（VHHs）和一种简化的酶联免疫吸附测定（ELISA）作为强大的新工具，用于获取单特异性试剂，以检测单个藻类细胞壁成分，并分离出具有特定细胞表面成分的藻类。
为开发新的微藻生物勘探工具，以帮助在环境样本中寻找具有相似细胞壁和细胞表面成分的藻类，我们制备了针对单细胞藻类莱茵衣藻细胞表面成分的单链骆驼科动物抗体。我们从骆驼科动物仅重链抗体中克隆了可变区结构域（VHHs），并在大肠杆菌中过量表达了这些带有标签的类单克隆抗体。利用这些VHHs，我们开发了一种准确、简便、低成本的ELISA方法，该方法使用活细胞作为天然构象抗原的来源，且检测过程耗时不到90分钟。已证明这种ELISA技术与使用细胞裂解物中的蛋白质且通常需要超过24小时才能完成的标准ELISA方法一样准确。在克隆的VHHs中，VHH B11对莱茵衣藻细胞表面表现出最高亲和力（EC50 < 1 nM）。采用活细胞ELISA程序检测自然环境水样中与莱茵衣藻具有相同细胞表面成分的藻类。此外，带有mCherry标签的VHH B11与荧光激活细胞分选（FACS）一起用于从相同环境样本中筛选假定的莱茵衣藻野生近缘种和其他绿藻纲的单个无菌分离株。
骆驼科动物抗体VHH结构域为检测藻类单个细胞壁成分以及从不同生态系统中选择具有特定细胞表面分子的藻类提供了一种高度特异性的工具。
