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Aptamer-Based Sandwich Assay for Measurement of Thymidine Kinase 1 in Serum of Cancerous Patients

机译:基于Aptamer的夹心测定,用于测量癌症患者血清中的胸苷激酶1

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摘要

Thymidine kinase 1 (TK1) is traditionally a serum biomarker that is elevated in the early stages of malignancies. The diagnostic and prognostic role of TK1 for screening and monitoring human malignancies has recently been investigated. Anti-human TK1 aptamers were selected through 12 iterative rounds of systematic evolution of ligands by exponential enrichment from a DNA library. The aptamer pool of round 12 was amplified, and the polymerase chain reaction product was cloned on the TA vector. Of the 85 colonies obtained, 52 were identified as positive clones. These aptamers were screened for TK1 with surface plasmon resonance, where apta37 and apta69 showed the highest affinity for TK1. The TK1_apta37 and TK1_apta69 aptamers were used in a sandwich assay platform and successfully detected TK1 in the concentration range of 54-3500 pg mL(-1). Clinical samples from 60 cancerous patients were also tested with this assay system and compared using the conventional antibody based enzyme-linked immunosorbent assay kit. The aptamer sandwich assay demonstrated a dynamic range for TK1 at clinically relevant serum levels, covering subpicogram per milliliter concentrations. The new approach offers a simple and robust method for detecting serum biomarkers that have low and moderate abundance. The results of this study demonstrate the screening capability of the aptamer sandwich assay platform and its potential applicability to the point-of-care testing system.
机译:胸苷激酶1(TK1)传统上是血清生物标志物,其在恶性肿瘤的早期阶段升高。 TK1对筛查和监测人类恶性肿瘤的诊断和预后作用最近已经调查过。通过指数富集来自DNA文库的指数富集来选择抗人类TK1适体通过12个迭代回合配体的系统演化。圆形12的适体池被扩增,并将聚合酶链反应产物克隆在TA载体上。在获得的85个菌落中,将52个被鉴定为阳性克隆。用表面等离子体共振筛选这些适体的TK1,其中APTA37和APTA69显示了TK1的最高亲和力。 TK1_APTA37和TK1_APTA69适体在夹层测定平台中使用,并在54-3500pg ml(-1)的浓度范围内成功地检测到TK1。来自60名癌症患者的临床样本也用该测定系统进行测试,并使用常规抗体基酶联免疫吸附试剂盒进行比较。 Aptamer Sandwich测定在临床相关的血清水平下表现出TK1的动态范围,覆盖每毫升浓度的亚本斑。新方法提供了一种简单且坚固的方法,用于检测具有低丰度和中等丰度的血清生物标志物。该研究的结果证明了适体夹心测定平台的筛选能力及其对护理点测试系统的潜在适用性。

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