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Creation of a Nanobody-Based Fluorescent Immunosensor Mini Q-body for Rapid Signal-On Detection of Small Hapten Methotrexate
ACS Sensors ( IF 8.2 ) Pub Date : 2020-11-10 , DOI: 10.1021/acssensors.0c01404 Akihito Inoue 1 , Yuki Ohmuro-Matsuyama 2 , Tetsuya Kitaguchi 2 , Hiroshi Ueda 2
ACS Sensors ( IF 8.2 ) Pub Date : 2020-11-10 , DOI: 10.1021/acssensors.0c01404 Akihito Inoue 1 , Yuki Ohmuro-Matsuyama 2 , Tetsuya Kitaguchi 2 , Hiroshi Ueda 2
Affiliation
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“Quenchbody (Q-body)” is a quench-based fluorescent biosensor labeled with a fluorescent dye near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules quickly by simply mixing with a sample. However, the development of Q-bodies using VHH-nanobodies derived from camelid heavy-chain antibodies has not been reported despite their favorable characteristics. Here, we report a “mini Q-body” that can detect the chemotherapy agent methotrexate (MTX) by using anti-MTX nanobody. Three kinds of constructs each encoding an N-terminal Cys-tag and anti-MTX VHH gene with a different length of linker (GGGS)n (n = 0, 2, and 4) between them were prepared followed by the expression in Escherichia coli and labeling with several dye maleimides. When the fluorescence intensities in the presence of varied MTX concentrations were measured, TAMRA-labeled nanobodies showed a higher response than ATTO520- or R6G-labeled ones. Especially, TAMRA C6-labeled mini Q-body with no linker showed the highest response of ∼6-fold and a low detection limit of 0.56 nM. When each Trp residue in the mini Q-body was mutated to address the quenching mechanism, the major role of Trp34 at CDR1 in quenching was revealed. Furthermore, the mini Q-body could detect MTX in 50% human serum with a low detection limit of 1.72 nM, showing its applicability to therapeutic drug monitoring. This study is expected to become the basis of the construction of highly responsive mini Q-bodies for sensitive detection of many molecules from small haptens to larger proteins, which will lead to broader applications such as point-of-care tests.
中文翻译：
基于纳米抗体的荧光免疫传感器迷你Q体的创建，用于小信号甲氨蝶呤的快速信号检测
“猝灭体（Q-body）”是基于猝灭的荧光生物传感器，其在抗体的抗原结合位点附近用荧光染料标记。Q体只需与样品混合即可快速检测出一系列靶分子。然而，用V Q-机构的发展HH -nanobodies从骆驼重链抗体衍生尚未见报道，尽管他们的良好特性。在这里，我们报告了一个“微型Q体”，可以通过使用抗MTX纳米抗体来检测化疗药物甲氨蝶呤（MTX）。制备了三种构建体，每种构建体编码N端Cys标签和抗MTX VHH基因，它们之间具有不同长度的连接子（GGGS）n（n = 0、2和4），然后在大肠杆菌中表达并用几种染料马来酰亚胺标记。当在不同的MTX浓度下测量荧光强度时，TAMRA标记的纳米抗体显示出比ATTO520或R6G标记的纳米抗体更高的响应。尤其是，没有接头的TAMRA C6标记的微型Q体显示出最高的响应约6倍，检测限低至0.56 nM。当微型Q体中的每个Trp残基突变以解决淬灭机理时，揭示了CDR1上Trp34在淬灭中的主要作用。此外，微型Q体可以在50％的人血清中检测MTX，检测限低至1.72 nM，显示了其在治疗药物监测中的适用性。这项研究有望成为构建高响应性迷你Q体的基础，该体能灵敏地检测从小半抗原到大蛋白的许多分子，
更新日期：2020-11-25
中文翻译：
基于纳米抗体的荧光免疫传感器迷你Q体的创建，用于小信号甲氨蝶呤的快速信号检测
“猝灭体（Q-body）”是基于猝灭的荧光生物传感器，其在抗体的抗原结合位点附近用荧光染料标记。Q体只需与样品混合即可快速检测出一系列靶分子。然而，用V Q-机构的发展HH -nanobodies从骆驼重链抗体衍生尚未见报道，尽管他们的良好特性。在这里，我们报告了一个“微型Q体”，可以通过使用抗MTX纳米抗体来检测化疗药物甲氨蝶呤（MTX）。制备了三种构建体，每种构建体编码N端Cys标签和抗MTX VHH基因，它们之间具有不同长度的连接子（GGGS）n（n = 0、2和4），然后在大肠杆菌中表达并用几种染料马来酰亚胺标记。当在不同的MTX浓度下测量荧光强度时，TAMRA标记的纳米抗体显示出比ATTO520或R6G标记的纳米抗体更高的响应。尤其是，没有接头的TAMRA C6标记的微型Q体显示出最高的响应约6倍，检测限低至0.56 nM。当微型Q体中的每个Trp残基突变以解决淬灭机理时，揭示了CDR1上Trp34在淬灭中的主要作用。此外，微型Q体可以在50％的人血清中检测MTX，检测限低至1.72 nM，显示了其在治疗药物监测中的适用性。这项研究有望成为构建高响应性迷你Q体的基础，该体能灵敏地检测从小半抗原到大蛋白的许多分子，
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