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Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon α2b
Journal of Pharmaceutical Analysis ( IF 6.1 ) Pub Date : 2021-07-08 , DOI: 10.1016/j.jpha.2021.07.003
Xi Qin 1 , Maoqin Duan 1 , Dening Pei 1 , Jian Lin 2 , Lan Wang 1 , Peng Zhou 2 , Wenrong Yao 1 , Ying Guo 1 , Xiang Li 1 , Lei Tao 1 , Youxue Ding 1 , Lan Liu 1 , Yong Zhou 1 , Chuncui Jia 1 , Chunming Rao 1 , Junzhi Wang 1, 3
Affiliation  

Recombinant human interferon α2b (rhIFNα2b) is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C. The current identification test for rhIFNα2b is complex. In this study, an anti-rhIFNα2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNα2b. RhIFNα2b was used to immunize an alpaca, which established a phage nanobody library. After five steps of enrichment, the nanobody I22, which specifically bound rhIFNα2b, was isolated and inserted into the prokaryotic expression vector pET28a. After subsequent purification, the physicochemical properties of the nanobody were determined. A semiquantitative detection and rapid identification assay of rhIFNα2b was developed using this novel nanobody. To develop a rapid test, the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips. The developed rhIFNα2b detection assay had a limit of detection of 1 μg/mL. The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products. The principle of this novel assay is generally applicable for the rapid testing of other commercial products, with a great potential for routine use in detecting counterfeit recombinant protein products.



中文翻译:


开发基于新型纳米抗体的侧流免疫层析试纸条快速检测重组人干扰素α2b



重组人干扰素α2b(rhIFNα2b)被广泛用作治疗乙型肝炎和丙型肝炎的抗病毒治疗剂。目前rhIFNα2b的鉴定测试很复杂。在这项研究中,发现了一种抗rhIFNα2b纳米抗体,并用于开发快速侧流试纸条以鉴定rhIFNα2b。使用RhIFNα2b免疫羊驼,建立了噬菌体纳米抗体库。经过五步富集,分离出特异性结合rhIFNα2b的纳米抗体I22,并将其插入原核表达载体pET28a中。经过后续纯化后,测定了纳米抗体的理化性质。使用这种新型纳米抗体开发了 rhIFNα2b 的半定量检测和快速鉴定方法。为了开发快速测试,将纳米抗体 I22 与胶体金偶联以生产侧流测试条。开发的 rhIFNα2b 检测方法的检测限为 1 μg/mL。 I22 的分离和侧流免疫层析测试条的成功构建证明了使用重组蛋白产品在侧流测试条上进行配体结合测定的可行性。这种新颖检测方法的原理普遍适用于其他商业产品的快速检测,在检测假冒重组蛋白产品的常规应用中具有巨大的潜力。

更新日期:2021-07-08
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