磁分离技术和纳米金比色法用于嗜碱性粒细胞活化试验研究

Basophil Activation Test Study Based on Magnetic Separation Technique and Gold Nanoparticle Colorimetry

嗜碱性粒细胞活化试验是一种安全、高效的诊断过敏疾病技术,成为临床体外诊断过敏疾病的主要方法之一。目前,嗜碱性粒细胞活化试验主要是利用流式细胞术对细胞进行检测,这种方法通常存在检测时间长、操作繁琐和成本高等缺点。针对这些问题,本研究开发了一种表面修饰HLA-DR抗体的磁珠分离体系,将其与过敏患者的外周血共孵育5 min后,利用磁性分离技术,筛选出表面含有HLA-DR标记物的细胞。测试结果表明,筛选后的嗜碱性粒细胞在全血中的细胞占比从0.2%提升到11.5%,嗜碱性粒细胞活化试验检测灵敏性得到了有效提高。进一步地,本研究构建了CD63适配体修饰的金纳米粒子检测体系,利用CD63适配体特异性识别活化后的嗜碱性粒细胞表面的CD63抗体,使金纳米粒子在嗜碱性粒细胞表面聚集,溶液颜色由红色转变为蓝紫色,实现对激活的嗜碱性粒细胞可视化比色检测,为临床开展嗜碱性粒细胞活化试验提供了新的方法。

Basophil activationtest(BAT)is a safe and efficient technique for the diagnosis of allergic diseases,which is one of the main methods for the clinical diagnosis of allergic diseases in vitro.Flow cytometry has been mainly used in BAT.However,this method showed some disadvantages,such as long detection time,cumbersome operation and high cost.To solve these problems,we developed a magnetic bead separation system with surface modified HLA-DR antibody.After co-incubation with peripheral blood of allergic patients for 5 min,cells with HLA-DR markers on the surface were seprated by magnetic separation technology.The results showed that the proportion of basophil cells in whole blood increased from 0.2%to 11.5%after screening,which effectively improved the sensitivity of BAT.Further,we developed a gold nanoparticle-based colorimetry with the CD63 aptamer modification on the surface of gold nanoparticles.Due to the specificity identification of CD63 aptamer with CD63 antibody after activation of basophils,the gold nanoparticles can gather together on the surface of basophils,thus resulting in the color changing from red to violet,which finally realized visual colorimetric detection of the activation of basophils.We herein believe our strategy will provide a new method for clinical BAT.