Traditional sandwich enzyme-linked immunosorbent assay (ELISA) using polyclonal and monoclonal antibodies as reagents presents several drawbacks, including limited amounts, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies can be easily expressed with different systems and fused with several tags in their tertiary structure by recombinant technology, thus offering an effective detection method for diagnostic purposes. Recently, the fenobody (ferritin-fused nanobody) and RANbody (nanobody-fused reporter) have been designed and derived from the nanobody for developing the diagnostic immunoassays. However, there was no report about developing the sandwich ELISA using the fenobody and RANbody as pairing reagents. A platform for developing a sandwich ELISA utilizing fenobody as the capture antibody and RANbody as the detection antibody was firstly designed in the study. Newcastle disease virus (NDV) was selected as the antigen, from which 13 NDV-specific nanobodies were screened from an immunized Bactrian camel. Then, 5 nanobodies were selected to produce fenobodies and RANbodies. The best pairing of fenobodies (NDV-fenobody-4, 800 ng/well) and RANbodies (NDV-RANbody-49, 1:10) was determined to develop the sandwich ELISA for detecting NDV. The detection limits of the assay were determined to be 22 of hemagglutination (HA) titers and 10 ng of purified NDV particles. Compared with two commercial assays, the developed assay shows higher sensitivity and specificity. Meanwhile, it exhibits 98.7% agreement with the HA test and can detect the reference NDV strains belonging to Class II but not Class I. In the presented study, the 13 anti-NDV nanobodies binding the NDV particles were first produced. Then, for the first time, the sandwich ELISA to detect the NDV in the different samples has been developed using the fenobody and RANbody as reagents derived from the nanobodies. Considering the rapidly increasing generation of nanobodies, the platform can reduce the cost of production for the sandwich ELISA and be universally used to develop assays for detecting other antigens.

中文翻译：

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基于Fenobody和RANbody的夹心酶联免疫吸附法检测新城疫病毒

使用多克隆和单克隆抗体作为试剂的传统夹心酶联免疫吸附测定（ELISA）存在几个缺点，包括数量有限，难以永久保存以及需要使用第二抗体。纳米抗体可通过重组技术轻松在不同系统中表达，并在其三级结构中与多个标签融合，从而为诊断目的提供了一种有效的检测方法。最近，已经设计并从纳米抗体衍生出了Fenobody（铁蛋白融合的纳米抗体）和RANbody（纳米抗体融合的报告基因），用于开发诊断性免疫测定。但是，没有关于使用fenobody和RANbody作为配对试剂开发夹心ELISA的报道。在研究中，首先设计了一个平台，该平台开发了一种以fenobody作为捕获抗体而RANbody作为检测抗体的夹心ELISA。选择新城疫病毒（NDV）作为抗原，从经过免疫的双峰驼中筛选出13种NDV特异性纳米抗体。然后，选择了5个纳米抗体来生产fenobody和RANbodies。确定了Fenobody（NDV-fenobody-4，800 ng /孔）和RANbodies（NDV-RANbody-49，1:10）的最佳配对，以开发用于检测NDV的夹心ELISA。测定的检出限确定为22个血凝（HA）滴度和10 ng纯化的NDV颗粒。与两种商业化的检测方法相比，开发的检测方法显示出更高的灵敏度和特异性。同时，它展出了98。7％的结果与HA测试一致，并且可以检测到属于II类而非I类的参考NDV菌株。在本研究中，首次生产了与NDV颗粒结合的13种抗NDV纳米抗体。然后，首次使用Fenobody和RANbody作为衍生自纳米抗体的试剂，开发了用于检测不同样品中NDV的夹心ELISA。考虑到纳米抗体的快速增长，该平台可降低夹心ELISA的生产成本，可普遍用于开发检测其他抗原的检测方法。使用fenobody和RANbody作为衍生自纳米抗体的试剂，已经开发出了用于检测不同样品中NDV的夹心ELISA。考虑到纳米抗体的快速增长，该平台可降低夹心ELISA的生产成本，可普遍用于开发检测其他抗原的检测方法。使用fenobody和RANbody作为衍生自纳米抗体的试剂，已经开发出了用于检测不同样品中NDV的夹心ELISA。考虑到纳米抗体的快速增长，该平台可降低夹心ELISA的生产成本，可普遍用于开发检测其他抗原的检测方法。