Screening, expression, and characterization of an anti-human oxidized low-density lipoprotein single-chain variable fragment
Screening, expression, and characterization of an anti-human oxidized low-density lipoprotein single-chain variable fragment
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DOI：
10.1016/j.jbiosc.2016.02.014
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发表时间：
2016-09-01
影响因子：
2.8
通讯作者：
Machida, Sachiko
中科院分区：
文献类型：
--
作者：
Kumano-Kuramochi, Miyuki;Fujimura, Takashi;Machida, Sachiko
血液循环中氧化低密度脂蛋白（OxLDL）水平的增加与动脉粥样硬化相关。已经报道了用于OxLDL的基于单克隆抗体的检测系统。我们确定了新的单链可变区片段（scFv）具有亲和力的人氧化低密度脂蛋白和相关配体。我们从非免疫的人脾mRNA中构建了scFv文库。通过生物淘选富集了两种类型（γ + κ和μ + λ）的scFv噬菌体文库，并鉴定了5个对OxLDL具有亲和力的scFv克隆。将对OxLDL具有最高亲和力的γ-κ 5单链抗体克隆到pET-22 b（+）中，并在大肠杆菌BL 21（DE 3）中表达。将在BL 21（DE 3）中表达为包涵体的γ κ 5重折叠并纯化。采用酶联免疫吸附试验（ELISA）分析γ κ 5的特异性和敏感性。γ κ 5 scFv显示出对OxLDL和乙酰化LDL的亲和力。γ κ 5对低浓度（1-2 μ g/mL）OxLDL的敏感性高于对AcLDL和LDL的敏感性。最后，我们开发了一种夹心ELISA使用γ κ 5和CTLD 14（凝集素样OxLDL受体-1配体识别区），它允许特异性检测OxLDL在低于0.1 μ g/mL的水平。我们的研究结果表明，γ κ 5 scFv是一个有前途的分子，用于检测修饰的LDL在非常低的浓度。(C)2016年，日本生物技术学会。All rights reserved.
Increased levels of oxidized low-density lipoprotein (OxLDL) in the blood circulation are correlated with atherosclerosis. Monoclonal antibody-based detection systems have been reported for OxLDL. We identified novel single-chain variable fragments (scFvs) having affinity for human OxLDL and related ligands. We constructed an scFv library from nonimmunized human spleen mRNA. Two types (gamma + kappa and mu + lambda) of scFv phage libraries were enriched by biopanning, and five scFv clones with affinity for OxLDL were identified. The gamma kappa 5 scFv, which showed the highest affinity for OxLDL, was cloned into pET-22b(+) and expressed in Escherichia coli BL21(DE3). gamma kappa 5, expressed as an inclusion body in BL21(DE3), was refolded and purified. The specificity and sensitivity of gamma kappa 5 were analyzed using enzyme-linked immunosorbent assays (ELISAs). The gamma kappa 5 scFv showed affinity for OxLDL and acetylated LDL. The sensitivity of gamma kappa 5 to low concentrations (1-2 mu g/mL) of OxLDL was higher than that to AcLDL and LDL. Finally, we developed a sandwich ELISA using gamma kappa 5 and CTLD14 (a lectin-like OxLDL receptor-1 ligand recognition region), which allowed specific detection of OxLDL at a level below 0.1 mu g/mL. Our results indicated that the gamma kappa 5 scFv was a promising molecule for the detection of modified LDL at very low concentrations. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.