Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?
Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?
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基于纳米抗体的人可溶性环氧化物水解酶检测免疫分析，使用聚合辣根过氧化物酶 (PolyHRP) 进行信号增强：PolyHRP 的重新发现？
DOI：
10.1021/acs.analchem.7b01247
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发表时间：
2017-06-06
影响因子：
7.4
通讯作者：
Ying Y
中科院分区：
文献类型：
--
作者：
Li D;Cui Y;Morisseau C;Gee SJ;Bever CS;Liu X;Wu J;Hammock BD;Ying Y
可溶性环氧化物水解酶（sEH）是治疗高血压、血管炎症、癌症、疼痛和多种心血管相关疾病的潜在药理靶点。重链抗体的可变结构域（称为单域抗体（sdAb），纳米体或VHH）具有体积小，稳定性高，易于遗传操作和连续制造的优点，使其成为免疫试剂的首选。在这项工作中，我们利用聚合辣根过氧化物酶（PolyHRP）来增强信号，开发了一种超灵敏的基于纳米体的人sEH免疫检测方法。在夹心酶联免疫吸附试验（ELISA）中，以羊驼抗人sEH的纳米抗体为检测抗体，以多克隆抗sEH为捕获抗体。以辣根过氧化物酶（HRP）标记的抗血凝素（HA）标记为示踪剂的传统夹心ELISA，边际灵敏度为0.0015光密度（OD）·mL/ng，检出限（LOD）为3.02 ng/mL。然而，引入PolyHRP作为示踪剂后，灵敏度提高了141倍（0.21 OD·mL/ng）， LOD降低了57倍（0.05 ng/mL）。系统比较了四种ELISA格式的三种不同示踪剂，证明了PolyHRP作为基于纳米体的免疫测定标记的压倒性优势。这种增强的sEH免疫分析进一步评估了对其他环氧化物水解酶的选择性和对人体组织匀浆样品中目标蛋白的检测。与酶活性法和Western blot法进行sEH检测的比较表明，sEH检测与免疫分析具有良好的相关性。这项工作证明了纳米体在利用PolyHRP检测sEH蛋白方面具有更高的竞争力。经过几十年的低调存在，PolyHRP在纳米体和其他基于亲和的方法上的潜力值得重新发现。
Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain, and multiple cardiovascular related diseases. A variable domain of the heavy chain antibody (termed single domain antibody (sdAb), nanobody, or VHH) possesses the advantages of small size, high stability, ease of genetic manipulation, and ability for continuous manufacture, making such nanobody a superior choice as an immunoreagent. In this work, we developed an ultrasensitive nanobody based immunoassay for human sEH detection using polymeric horseradish peroxidase (PolyHRP) for signal enhancement. Llama nanobodies against human sEH were used as the detection antibody in sandwich enzyme linked immunosorbent assays (ELISA) with polyclonal anti-sEH as the capture antibody. A conventional sandwich ELISA using a horseradish peroxidase (HRP) labeled anti-hemeagglutinin (HA) tag as the tracer showed a marginal sensitivity (0.0015 optical density (OD)·mL/ng) and limit of detection (LOD) of 3.02 ng/mL. However, the introduction of the PolyHRP as the tracer demonstrated a 141-fold increase in the sensitivity (0.21 OD·mL/ng) and 57-fold decrease in LOD (0.05 ng/mL). Systematic comparison of three different tracers in four ELISA formats demonstrated the overwhelming advantage of PolyHRP as a label for nanobody based immunoassay. This enhanced sEH immunoassay was further evaluated in terms of selectivity against other epoxide hydrolases and detection of the target protein in human tissue homogenate samples. Comparison with an enzyme activity based assay and a Western blot for sEH detection reveals good correlation with the immunoassay. This work demonstrates increased competiveness of nanobodies for practical sEH protein detection utilizing PolyHRP. It is worthwhile to rediscover the promising potential of PolyHRP in nanobody and other affinity based methods after its low-profile existence for decades.
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影响因子：
17.1
作者：
Mani, Vigneshwaran;Chikkaveeraiah, Bhaskara V.;Patel, Vyomesh;Gutkind, J. Silvio;Rusling, James F.
通讯作者：
Rusling, James F.
影响因子：
7.4
作者：
Rossotti MA;Pirez M;Gonzalez-Techera A;Cui Y;Bever CS;Lee KS;Morisseau C;Leizagoyen C;Gee S;Hammock BD;González-Sapienza G
通讯作者：
González-Sapienza G
影响因子：
7.4
作者：
Wang, Jia;Majkova, Zuzana;Bever, Candace R. S.;Yang, Jun;Gee, Shirley J.;Li, Ji;Xu, Ting;Hammock, Bruce D.
通讯作者：
Hammock, Bruce D.
影响因子：
7.4
作者：
Kim, Hee-Joo;McCoy, Mark R.;Majkova, Zuzana;Dechant, Julie E.;Gee, Shirley J.;Tabares-da Rosa, Sofia;Gonzalez-Sapienza, Gualberto G.;Hammock, Bruce D.
通讯作者：
Hammock, Bruce D.
影响因子：
7.4
作者：
Ambrosi, Adriano;Airo, Federico;Merkoci, Arben
通讯作者：
Merkoci, Arben