开发用于快速定量检测小分子化合物的横向流动免疫色谱带

Published: November 13, 2021
doi:

Abstract

基于膜的横向流动免疫色谱条(ICSs)是低成本自我诊断的有用工具,已有效地应用于毒素、生理指标和临床生物标志物检测。在此协议中,我们提供了开发快速、敏感和定量横向流动免疫分析(使用 AuNPs 作为标记和 mAbs 作为探头)的步骤的详细描述。该过程描述了胶体黄金的制备和定性、AuNP-mAb结合体的合成、免疫色谱条的组装以及检测的方法学研究。结果表明,最终条带可进一步用于小分子的快速、方便的自我诊断,为快速、准确地分析生理和生物指标提供了替代工具。

Introduction

基于膜的横向流动免疫色谱条 (ICS) 是低成本和快速检测的有用工具。硝基纤维素膜作为载体,胶体金作为免疫色谱快速诊断试剂的标志,是最常用的POCT(护理点测试)方法,项目测试范围更广。从他们最初在怀孕期间监测的应用,他们的使用已扩大到监测血液凝固状态1,2,心肌损伤3,兽药4,农药残留5,传染病6和药物浓度。更多的样本类型可以评估,包括尿液,唾液,全血,血清和其他体液7,8,9。

近年来,在疾病诊断中,已经开发出许多用于检测生物标志物的新检测方法,包括 HPLC、UPLC、LC-MS 和 ELISA,它们敏感、准确、可信和具体。然而,这些方法需要复杂的仪器,复杂的预处理和耗时的治疗9。因此,制定一种更快速、更方便的护理点诊断策略,对药用活性化合物进行自我和实时检测,是当务急。

ICS 的流行,特别是对于常见的测试,是由其易用性驱动的,因为它们不需要专业人员或精心设计的工具设置12。换句话说,没有接受过特殊训练的人可以操作条形或自考13。测试结果可在5分钟内获得,这意味着可用于现场检查14。此外,根据我们的计算,条的成本可能低于1元15,这意味着测试是便宜的推广16。因此,ICS 是一种相对准确、简单、廉价的一次性设备。基于胶体金17、18的ICS也应用于COVID-19的快速检测。

ICS 的原则可分为三明治 ICS 和具有竞争力的 ICS。 图1A 是三明治ICS的示意图,主要用于检测大分子物质,如蛋白质,包括肿瘤标记物、炎症因子和人类胆汁性腺激素(HCG,早孕抗原)。在这种方法中,使用针对抗原不同表位的配对抗体,并将捕获抗体作为测试线干燥在NC膜上。标记的抗体在结合垫上干燥,二级抗体用作控制线。

图1B 是竞争ICS的示意图,主要用于检测小分子物质(MWCO<2000 da)。涂层抗原固定在NC膜上作为测试线,标记的抗体在结合垫上干燥。在检测过程中,样品和标记的抗体在毛细管作用经检测线,涂层抗原在样品中具有竞争力地结合自由抗原,并在检测线上形成红色。

最近,我们描述了单克隆抗体生成对天然产品19的程序。在这项工作中,我们开发了一种新的横向流动免疫分析基于准备的抗SSD mA20 进行快速、现场检测。结果表明,免疫色谱检测是检测天然产品衍生化合物不可或缺的便捷工具。

Figure 1
图1 免疫色谱测定(A)三明治免疫色谱测试条的示意图图。(B) 间接竞争免疫色谱测试条。这个数字已经从张等人修改,2018年21日。请单击此处查看此图的较大版本。

Protocol

本研究的所有程序均获北京中医药大学伦理审查委员会批准(批准号2017BZYYL00120)。 1. 胶体黄金的制备和定性 注:用于胶体黄金合成, 由于胶体金容易吸附在容器内壁上,容易被杂质沉淀,因此应彻底清洗和浸泡在酸(40 mL蒸馏水、360mL浓缩硫酸、20克二色二铬钾)中,或进行表面钝化处理。使用柠檬酸还原法合成胶体金。 打开磁搅?…

Representative Results

胶体黄金的特征准备好的胶体黄金溶液是克拉雷特红色。TEM 分析用于确定 AuNPs 的形态和形状(图 2A-D)。图2A和图2B显示,粒子呈多面体形状,分布均匀。发现 AuNPs 的平均直径约为 14 nm(图 2C)。AuNPs 的高分辨率 TEM (HRTEM) 图像显示在图 2D和<…

Discussion

在这项工作中,我们提出了针对天然产品衍生小分子制备 mAb 的协议。已经概述了程序中需要注意的基本步骤和事项,我们以小分子SSD为例证明了该协议的效用。代表性数据中显示了示例光谱、TEM 图像、定量结果和方法研究。因此,我们已经证明,在这里提出的胶体黄金生产、AuNP-mAb结合和脱衣装配策略,创造了一种可用于快速检测目标分子的新型免疫分析。

免疫色谱条易于…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了中央直属高等学校基础研究经费专项资金的支持。我们感谢北京中医药大学经典处方基础研究团队的支持。

Materials

Chloroauric acid solution (HAuCl4) Tianjin Fu Chen Chemical Reagents Factory JY-SJ102
bovine serum albumin AMRESCO 332
centrifuge tube 15 mL Corning 430645
centrifuge tube 50 mL Corning 430828
ELISA plates, 96 well NUNC 655101
Filter paper Sinopharm H5072
Glass fibre membranes Jieyi XQ-Y6
goat-anti-mouse IgG antibody applygen C1308
Nitrocellulose membranes Millipore millipore 180
ovalbumin Beijing BIODEE 5008-25g
PEG20000 Sigma Aldrich RNBC6325
Pipette 10mL COSTAR 4488
Pipette 25mL FALCON 357525
semi-rigid PVC sheets Jieyi JY-C104
Sodium citrate Beijing Chemical Works C1034
sodium periodate Sinopharm Chemical BW-G0008
Sulfo-GMBS Perbio Science Germany 22324
TipOne Tips 1,000 µL Starlab S1111-2021
Tris-HCl Solarbio 77-86-1
TWEEN 20 Solarbio 9005-64-5

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Cite This Article
Zhang, Y., Cao, P., Lu, F., Cheng, J., Qu, H. Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds. J. Vis. Exp. (177), e62754, doi:10.3791/62754 (2021).

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