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基于蛋白质微阵列的诊断芯片的开发,模拟皮肤点刺试验用于过敏诊断。

Development of a protein microarray-based diagnostic chip mimicking the skin prick test for allergy diagnosis.

机构信息

Molecular Therapeutics and Formulation Division, School of Pharmacy, University of Nottingham, Nottingham, UK.

Advanced Materials and Healthcare Technologies Division, School of Pharmacy, University of Nottingham, Nottingham, UK.

出版信息

Sci Rep. 2020 Oct 23;10(1):18208. doi: 10.1038/s41598-020-75226-y.

Abstract

Protein microarrays have been successfully used for detection of allergen-specific IgE in patient sera. Here, we demonstrate proof-of-concept of a solid-phase technique coupling the high-throughput potential of protein microarrays with the biologically relevant readout provided by IgE reporter cells, creating a novel allergic sensitization detection system. Three proteins (κ-casein, timothy grass pollen extract, polyclonal anti-human IgE) were printed onto three different polymer-coated surfaces (aldehyde-, epoxy- and NHS ester-coated). ToF-SIMs analysis was performed to assess printed protein stability and retention during washing steps. NFAT-DsRed rat basophil leukemia cell attachment and retention during washing steps was assessed after treatment with various extracellular matrix proteins. NFAT-DsRed IgE reporter cells were sensitized with serum of an allergic donor, incubated on the printed slides, and cell activation determined using a microarray laser scanner. NFAT DsRed IgE reporter cell binding was significantly increased on all polymer surfaces after incubation with fibronectin and vitronectin, but not collagen or laminin. All surfaces supported printed protein stability during washing procedure, with epoxy- and NHS ester-coated surfaces showing best protein retention. Cell activation was significantly higher in NHS ester-coated slides after timothy grass pollen extract stimulation appearing a suitable substrate for further development of an automated allergy diagnosis system.

摘要

蛋白质微阵列已成功用于检测患者血清中的过敏原特异性 IgE。在这里,我们证明了一种固相技术的概念验证,该技术将蛋白质微阵列的高通量潜力与 IgE 报告细胞提供的生物学相关读数相结合,创建了一种新型的过敏致敏检测系统。将三种蛋白质(κ-酪蛋白、梯牧草花粉提取物、多克隆抗人 IgE)打印到三种不同的聚合物涂层表面(醛基、环氧基和 NHS 酯基)上。进行 ToF-SIMS 分析以评估在洗涤步骤中打印蛋白质的稳定性和保留情况。在用各种细胞外基质蛋白处理后,评估了 NFAT-DsRed 大鼠嗜碱性白血病细胞在洗涤步骤中的附着和保留情况。用过敏供体的血清敏化 NFAT-DsRed IgE 报告细胞,在打印的载玻片上孵育,并使用微阵列激光扫描仪测定细胞激活。NFAT DsRed IgE 报告细胞的结合在与纤连蛋白和 vitronectin 孵育后,在所有聚合物表面上均显著增加,但与胶原蛋白或层粘连蛋白没有增加。所有表面在洗涤过程中均支持打印蛋白质的稳定性,其中环氧基和 NHS 酯基涂层显示出最佳的蛋白质保留。在用梯牧草花粉提取物刺激后,NHS 酯涂层载玻片上的细胞激活显著增加,这似乎是进一步开发自动化过敏诊断系统的合适基质。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f40b/7584649/b52af10a508d/41598_2020_75226_Fig1_HTML.jpg

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