The continuous spread and evolution of severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) necessitate the development of convenient and rapid detection methods. In this study, we developed a fluorescence enzyme immunoassay (FEIA) based on a nanobody (Nb)–alkaline phosphatase (ALP) fusion protein for detection of SARS-CoV-2 spike protein. The genetically modified anti-SARS-CoV-2 S-RBD Nb, Nb61, gene was fused with the ALP gene sequences via a flexible linker. Recombinant cloning was used to yield a recombinant prokaryotic expression plasmid, Nb61-ALP-His. The Nb61-ALP-His construct was transformed into E. coli BL21(DE3) and expressed in bacteria. Both Nb61 properties and ALP enzymatic activity were validated by colorimetric and fluorometric analysis. FEIA was optimized and established on the basis of the Nb61-ALP fusion protein. The detection limit of the FEIA was 3.18 ng/mL, with a linear range of 1.9–62.5 ng/mL. Comparison with a commercial kit indicated the reliability of the Nb61-ALP fusion-protein-based FEIA for monitoring the SARS-CoV-2 spike protein. This study highlights the potential of Nb-based enzyme immunoassays as a valuable tool for the rapid and accurate detection of SARS-CoV-2.

中文翻译：

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开发纳米抗体-碱性磷酸酶融合蛋白，用于在荧光酶免疫测定中检测 SARS-CoV-2 刺突蛋白


严重急性呼吸道症状冠状病毒 2 （SARS-CoV-2） 的持续传播和进化需要开发方便和快速的检测方法。在这项研究中，我们开发了一种基于纳米抗体 （Nb）-碱性磷酸酶 （ALP） 融合蛋白的荧光酶免疫测定法 （FEIA），用于检测 SARS-CoV-2 刺突蛋白。转基因抗 SARS-CoV-2 S-RBD Nb， Nb61 基因通过柔性接头与 ALP 基因序列融合。使用重组克隆产生重组原核表达质粒 Nb61-ALP-His。将 Nb61-ALP-His 构建体转化到大肠杆菌 BL21 （DE3） 中并在细菌中表达。Nb61 特性和 ALP 酶活性均通过比色和荧光分析进行验证。FEIA 是在 Nb61-ALP 融合蛋白的基础上优化和建立的。FEIA 的检测限为 3.18 ng/mL，线性范围为 1.9–62.5 ng/mL。与商业试剂盒的比较表明，基于 Nb61-ALP 融合蛋白的 FEIA 在监测 SARS-CoV-2 刺突蛋白方面的可靠性。本研究强调了基于 Nb 的酶免疫测定法作为快速准确检测 SARS-CoV-2 的宝贵工具的潜力。