用于检测热带屋尘螨苏氏嗜木螨的金纳米颗粒标记夹心型侧向流动免疫分析试剂盒的研制

Development of a Gold Nanoparticle-labeled Sandwich Format Lateral Flow Immunoassay Kit for the Detection of Tropical House Dust Mite Suidasia pontifica.

作者信息

Ngu Mark Angelo V, Bergantin Jose H, Ramos John Donnie A

机构信息

The Graduate School, University of Santo Tomas, Espana Boulevard, Sampaloc 1015, Manila, Philippines.

College of Science, University of Santo Tomas, España Boulevard, Sampaloc 1015, Manila, Philippines.

出版信息

Protein Pept Lett. 2019;26(5):357-363. doi: 10.2174/0929866526666190212164751.

DOI:10.2174/0929866526666190212164751

PMID:30760184

Abstract

BACKGROUND

The house dust mite Suidasia pontifica (Sp) is an important source of allergens in tropical regions that trigger IgE-mediated allergic reactions such as allergic asthma, atopic dermatitis and allergic rhinitis. Detection of Sp-specific proteins are important in the management and prevention of allergic diseases.

OBJECTIVE

The study aimed to provide a proof of concept for a gold nanoparticle-labeled sandwich format Lateral Flow Immunoassay (LFIA) kit for the detection of Sp-specific proteins.

METHODS

Protein A chromatography-purified rabbit anti-Sp polyclonal antibodies were labeled with gold nanoparticles (AuNP) synthesized from chloroauric acid using the citrate reduction method, then dispensed on a glass fiber pad. Unlabeled antibodies and anti-rabbit IgG were immobilized onto nitrocellulose membrane as test line and control line respectively. Cellulose fiber pad, glass fiber, and the nitrocellulose membrane pad were then assembled as LFIA kit.

RESULTS

Protein-A affinity chromatography purification with pre-concentration yielded 1.45 mg/mL of anti-Sp polyclonal antibodies. Synthesized AuNPs with ~20 nm sizes observed under transmission electron microscope were used for antibody conjugation at an optimal pH of 8.5 (borate buffer) and an optimal ratio of 10 µ L 50µg/mL antibody:100 µ L AuNP. Optimal color intensity and fastest migration time were observed with the treatment of 0.05% Tween20 and 10% sucrose in the conjugate pads; 5% BSA and 0.05% Tween20 in the sample pads, and 1% BSA in the test pads. The limit of detection of the LFIA Sp-specific proteins is 0.076 µg/mL. The sensitivity of the Sp LFIA kit is 83% while the specificity is 100%.

CONCLUSION

This is the first report of a prototype for a cost-effective, rapid, and equipment-free detection of the house dust mite Suidasia pontifica.

摘要

背景

热带地区的屋尘螨苏伊氏钝绥螨（Sp）是引发IgE介导的过敏反应（如过敏性哮喘、特应性皮炎和过敏性鼻炎）的重要过敏原来源。检测Sp特异性蛋白对过敏性疾病的管理和预防至关重要。

目的

本研究旨在为用于检测Sp特异性蛋白的金纳米颗粒标记夹心式侧向流动免疫分析（LFIA）试剂盒提供概念验证。

方法

用蛋白A层析法纯化的兔抗Sp多克隆抗体，用柠檬酸还原法从氯金酸合成的金纳米颗粒（AuNP）标记，然后点样于玻璃纤维垫上。未标记的抗体和抗兔IgG分别固定在硝酸纤维素膜上作为检测线和对照线。然后将纤维素纤维垫、玻璃纤维和硝酸纤维素膜垫组装成LFIA试剂盒。

结果

经预浓缩的蛋白A亲和层析纯化得到浓度为1.45 mg/mL的抗Sp多克隆抗体。在透射电子显微镜下观察到合成的尺寸约为20 nm的AuNP，在最佳pH值8.5（硼酸盐缓冲液）和最佳比例10 µL 50µg/mL抗体:100 µL AuNP下用于抗体偶联。在结合垫中用0.05%吐温20和10%蔗糖处理；在样品垫中用5%牛血清白蛋白和0.05%吐温20处理，在检测垫中用1%牛血清白蛋白处理时，观察到最佳颜色强度和最快迁移时间。LFIA检测Sp特异性蛋白的检测限为0.076 µg/mL。Sp LFIA试剂盒的灵敏度为83%，特异性为100%。