Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology, Ministry of Agriculture, Shaanxi, 712100, China.
J Nanobiotechnology. 2020 Mar 14;18(1):44. doi: 10.1186/s12951-020-00598-2.
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 2 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.
传统的夹心酶联免疫吸附测定（ELISA）使用多克隆和单克隆抗体作为试剂存在几个缺点，包括数量有限、难以永久储存以及需要使用二抗。纳米抗体可以通过重组技术在不同的系统中轻松表达，并在其三级结构中融合多个标签，从而为诊断目的提供有效的检测方法。最近，已经设计并衍生出 fenobody（铁蛋白融合纳米抗体）和 RANbody（纳米抗体融合报告物），用于开发诊断免疫测定。然而，目前尚无关于使用 fenobody 和 RANbody 作为配对试剂开发夹心 ELISA 的报道。
本研究首次设计了一种利用 fenobody 作为捕获抗体、RANbody 作为检测抗体开发夹心 ELISA 的平台。选择新城疫病毒（NDV）作为抗原，从免疫的双峰驼中筛选出 13 种 NDV 特异性纳米抗体。然后，选择 5 种纳米抗体来产生 fenobody 和 RANbody。确定 fenobodies（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。该测定的检测限确定为 2 个血凝（HA）效价和 10 ng 纯化的 NDV 颗粒。与两种商业测定方法相比，该方法显示出更高的灵敏度和特异性。同时，它与 HA 试验的一致性达到 98.7%，并且可以检测属于 II 类而不属于 I 类的参考 NDV 株。
在本研究中，首次生产了结合 NDV 颗粒的 13 种抗 NDV 纳米抗体。然后，首次使用 fenobody 和 RANbody 作为试剂，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生成，该平台可以降低夹心 ELISA 的生产成本，并可普遍用于开发检测其他抗原的测定。
