基于单克隆抗体的电化学免疫传感器用于猪流行性腹泻病毒检测

Abstract

This study reports the development of a novel and highly sensitive electrochemical immunosensor for detecting porcine epidemic diarrhea virus (PEDV). To this end, a unique PEDV spike protein–specific monoclonal antibody (mAb) was generated and evaluated using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), western blot (WB), and flow cytometry. The mAb, classified as an IgG1 subtype in the light chain, recognizes a linear epitope of the PEDV S protein ( 696 SVTPCSFSEQAAYVDDDIV 715 ). Further analysis showed that this epitope (amino acids 696–715) is localized on the surface of the PEDV S protein in the 3D structure. The antibody was then immobilized on the immunosensor to specifically recognize PEDV. A screen-printed electrode (SPE) modified with gold nanoparticles (AuNPs) was prepared and coupled to the PEDV mAb 1G10 via protein A (Pro A) to construct a new PEDV immunosensor. PEDV concentration was quantified by measuring changes in charge transfer resistance after antigen–antibody binding. The proposed immunosensor demonstrated excellent repeatability, stability, and specificity, and was successfully applied to detect PEDV in real samples. Key points •  Identification of a novel linear epitope ( 696 S-V 715 ) on the PEDV spike protein. •  Development of a highly sensitive electrochemical immunosensor for PEDV detection. •  The biosensor accurately detected PEDV in clinical pig manure samples.