Ding Yuanyuan, Li Xiaoqian, Gao Qingpeng, Dong Xinyan, Kong Liyun, Han Shengli, Zhang Tao, He Langchong
School of Pharmacy, Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, China.
School of Public Health, Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, China.
Anal Biochem. 2021 Nov 15;633:114392. doi: 10.1016/j.ab.2021.114392. Epub 2021 Sep 28.
Mas-related G-protein-coupled receptor X2 (MRGPRX2) has recently been reported to be associated with anaphylaxis. Detection of MRGPRX2 levels in human peripheral blood might serve as a powerful tool for predicting the predisposition of patients to anaphylactic reactions. For rapid measurement of MRGPRX2, we established a paper-based double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody and horseradish peroxidase (HRP)-labelled rabbit polyclonal antibody as capture antibody and detection antibody, respectively. We avoided chemical functionalization of the cellulose paper by introducing bovine serum albumin (BSA) to provide COOH and NH groups for covalent immobilization of the capture antibody. Through amide condensation, a two-layer immobilization strategy was applied with BSA-BSA and BSA-capture antibody networks as the first and second layers, respectively. This strategy improved the quantity, activity and stability of the immobilized antibody. We then established a paper-based ELISA to detect MRGPRX2 in human peripheral blood. Our method is less laborious, easier to implement, and more cost-effective than conventional ELISA, while offering similar sensitivity, specificity, and accuracy. Therefore, it could serve as an innovative clinical point-of-care diagnostic tool, especially in areas that lack advanced clinical equipment.
最近有报道称，与 Mas 相关的 G 蛋白偶联受体 X2（MRGPRX2）与过敏反应有关。检测人外周血中的 MRGPRX2 水平可能成为预测患者过敏反应易感性的有力工具。为了快速检测 MRGPRX2，我们建立了一种基于纸的双抗体夹心酶联免疫吸附测定法（ELISA），分别使用小鼠单克隆抗体和辣根过氧化物酶（HRP）标记的兔多克隆抗体作为捕获抗体和检测抗体。我们通过引入牛血清白蛋白（BSA）来避免纤维素纸的化学功能化，以提供用于捕获抗体共价固定的羧基和氨基。通过酰胺缩合，应用了一种两层固定策略，分别以 BSA-BSA 和 BSA-捕获抗体网络作为第一层和第二层。该策略提高了固定化抗体的数量、活性和稳定性。然后我们建立了一种基于纸的 ELISA 来检测人外周血中的 MRGPRX2。我们的方法比传统 ELISA 省力、更易于实施且成本效益更高，同时具有相似的灵敏度、特异性和准确性。因此，它可以作为一种创新的即时临床诊断工具，特别是在缺乏先进临床设备的地区。
