Development of a Nanobody-Based Lateral Flow Immunoassay for Detection of Human Norovirus.
Development of a Nanobody-Based Lateral Flow Immunoassay for Detection of Human Norovirus.
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DOI：
10.1128/msphere.00219-16
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发表时间：
2016-09
期刊：
影响因子：
4.8
通讯作者：
Hansman GS
中科院分区：
文献类型：
--
作者：
Doerflinger SY;Tabatabai J;Schnitzler P;Farah C;Rameil S;Sander P;Koromyslova A;Hansman GS
我们先前鉴定了与诺如病毒衣壳上的高度保守区域结合的纳米抗体（称为Nano-85）。在该研究中，纳米抗体被生物素化并与金缀合用于侧流免疫测定（称为Nano-IC）。我们表明，纳米IC检测能够检测至少四种抗原性不同的GII基因型，包括新出现的GII。在临床环境中，Nano-IC测定的灵敏度与其他市售侧流系统相当。Nano-IC方法能够在约5分钟内产生结果，这使得该方法在需要快速诊断的环境中非常有用，例如游轮爆发和老年护理设施。Nano-IC测定与基于抗体的IC方法相比具有几个优点：例如，纳米抗体可以容易地大量生产，它们通常比常规抗体更稳定，并且纳米抗体结合位点可以通过X射线晶体学容易地获得。人类诺如病毒是急性胃肠炎暴发的主要原因。这些病毒通常通过分子方法检测，包括逆转录酶PCR（RT-PCR）和酶联免疫吸附测定（ELISA）。人类诺如病毒在遗传和抗原上是多样的，具有两个主要基因组，其进一步细分为超过40种不同的基因型。在过去十年中，基因组2基因型4（GII.4）在大多数国家占主导地位，但最近，属于GII.17的病毒在一些国家的流行率有所增加。发现许多市售ELISA和侧流免疫测定对GII.17病毒的敏感性较低，表明这些方法中使用的抗体可能不具有高水平的交叉反应性。在这项研究中，我们开发了一种快速的基于纳米抗体的侧流免疫分析（纳米免疫色谱[Nano-IC]），用于检测临床标本中的人诺如病毒。Nano-IC检测试剂盒检测了来自两个GII.4诺如病毒簇的病毒体，其中包括当前的优势毒株和一种新型变异毒株。Nano-IC方法对暴发疫情标本的敏感性为80%，特异性为86%。通过该方法可以检测代表四种基因型（GII.4、GII.10、GII.12和GII.17）的诺如病毒病毒样颗粒（VLP），证明了其在临床筛查中的潜力。然而，需要对Nano-IC方法进行进一步修改以提高这种灵敏度，这可以通过向系统中添加其他广泛反应性的纳米抗体来实现。我们先前鉴定了一种纳米抗体（称为Nano-85），其结合到诺如病毒衣壳上的高度保守区域。在该研究中，纳米抗体被生物素化并与金缀合用于侧流免疫测定（称为Nano-IC）。我们表明，纳米IC检测能够检测至少四种抗原性不同的GII基因型，包括新出现的GII。在临床环境中，Nano-IC测定的灵敏度与其他市售侧流系统相当。Nano-IC方法能够在约5分钟内产生结果，这使得该方法在需要快速诊断的环境中非常有用，例如游轮爆发和老年护理设施。Nano-IC测定与基于抗体的IC方法相比具有几个优点：例如，纳米抗体可以容易地大量生产，它们通常比常规抗体更稳定，并且纳米抗体结合位点可以通过X射线晶体学容易地获得。
We previously identified a Nanobody (termed Nano-85) that bound to a highly conserved region on the norovirus capsid. In this study, the Nanobody was biotinylated and gold conjugated for a lateral flow immunoassay (termed Nano-IC). We showed that the Nano-IC assay was capable of detecting at least four antigenically distinct GII genotypes, including the newly emerging GII.17. In the clinical setting, the Nano-IC assay had sensitivities equivalent to other commercially available lateral flow systems. The Nano-IC method was capable of producing results in ~5 min, which makes this method useful in settings that require rapid diagnosis, such as cruise ship outbreaks and elder care facilities. The Nano-IC assay has several advantages over antibody-based IC methods: for example, Nanobodies can be readily produced in large quantities, they are generally more stable than conventional antibodies, and the Nanobody binding sites can be easily obtained by X-ray crystallography. Human noroviruses are the dominant cause of outbreaks of acute gastroenteritis. These viruses are usually detected by molecular methods, including reverse transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Human noroviruses are genetically and antigenically diverse, with two main genogroups that are further subdivided into over 40 different genotypes. During the past decade, genogroup 2 genotype 4 (GII.4) has dominated in most countries, but recently, viruses belonging to GII.17 have increased in prevalence in a number of countries. A number of commercially available ELISAs and lateral flow immunoassays were found to have lower sensitivities to the GII.17 viruses, indicating that the antibodies used in these methods may not have a high level of cross-reactivity. In this study, we developed a rapid Nanobody-based lateral flow immunoassay (Nano-immunochromatography [Nano-IC]) for the detection of human norovirus in clinical specimens. The Nano-IC assay detected virions from two GII.4 norovirus clusters, which included the current dominant strain and a novel variant strain. The Nano-IC method had a sensitivity of 80% and specificity of 86% for outbreak specimens. Norovirus virus-like particles (VLPs) representing four genotypes (GII.4, GII.10, GII.12, and GII.17) could be detected by this method, demonstrating the potential in clinical screening. However, further modifications to the Nano-IC method are needed in order to improve this sensitivity, which may be achieved by the addition of other broadly reactive Nanobodies to the system. IMPORTANCE We previously identified a Nanobody (termed Nano-85) that bound to a highly conserved region on the norovirus capsid. In this study, the Nanobody was biotinylated and gold conjugated for a lateral flow immunoassay (termed Nano-IC). We showed that the Nano-IC assay was capable of detecting at least four antigenically distinct GII genotypes, including the newly emerging GII.17. In the clinical setting, the Nano-IC assay had sensitivities equivalent to other commercially available lateral flow systems. The Nano-IC method was capable of producing results in ~5 min, which makes this method useful in settings that require rapid diagnosis, such as cruise ship outbreaks and elder care facilities. The Nano-IC assay has several advantages over antibody-based IC methods: for example, Nanobodies can be readily produced in large quantities, they are generally more stable than conventional antibodies, and the Nanobody binding sites can be easily obtained by X-ray crystallography.