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首页> 外文期刊>Harmful Algae >In vitro isolation of nanobodies for selective Alexandrium minutum recognition: A model for convenient development of dedicated immuno- reagents to study and diagnostic toxic unicellular algae
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In vitro isolation of nanobodies for selective Alexandrium minutum recognition: A model for convenient development of dedicated immuno- reagents to study and diagnostic toxic unicellular algae

机译：用于选择性亚历山大藻识别的纳米抗体的体外分离：方便开发专用免疫试剂以研究和诊断有毒单细胞藻类的模型

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摘要

At the present, the identification of planktonic species in coastal water is still a time intensive process performed by highly trained personnel that relies either on qPCR or on light microscopy observation and in vitro culturing. Furthermore, the increasing danger represented by Harmful Algal Blooms (HABs) inside phytoplankton community and the recent implementation of the legislation on ballast water management to prevent the introduction of HABs and NIS (Non Indigenous Species) urge the development of faster and reliable diagnostic methods. Immuno-based approaches could fulfil this need provided that the costs for antibody selection and production will be reduced.In this work it is demonstrated for the first time the feasibility to recover nanobodies (VHHs) selective for native surface epitopes of Alexandrium minutum by direct whole cell bio-panning using a pre-immune phage display library. The recombinant nature of VHHs enabled their rapid engineering into eGFP fluorescent reagents (fluobodies) that were produced recombinantly in bacteria and are directly suitable for fluorescence microscopy and flow cytometry. Immune-detection identified also cysts and anti-Alexandrium fluobodies showed no cross-reactivity with indigenous not-toxic phytoplankton microalgae belonging to different geni. The fluobodies were able to bind selectively to the target cells in both fixed and fresh samples with minimal processing.
机译：目前，在沿海水域中确定浮游生物种类仍然是由训练有素的人员进行的耗时的过程，其依赖于qPCR或光学显微镜观察和体外培养。此外，浮游植物群落内部有害藻华（HABs）所代表的危险性日益增加，以及最近实施的压载水管理立法以防止引入HABs和NIS（非土著物种），促使人们开发出更快，更可靠的诊断方法。只要减少抗体选择和生产的成本，基于免疫的方法就可以满足这一需求。在这项工作中，这首次证明了通过直接整体回收对亚历山大草天然表位有选择性的纳米抗体（VHH）的可行性。使用免疫前噬菌体展示文库进行细胞生物学淘选。 VHHs的重组性质使其能够快速工程化为在细菌中重组产生的eGFP荧光试剂（荧光体），直接适用于荧光显微镜和流式细胞术。免疫检测还鉴定出囊肿和抗亚历山大藻荧光体与属于不同属的本地无毒浮游植物微藻没有交叉反应。荧光体能够以最少的处理选择性结合固定样品和新鲜样品中的靶细胞。

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