A multiplex fluorescent lateral flow immunoassay based on europium nanoparticles for simultaneous detection of Helicobacter pylori infection and virulence-associated antibodies.

Helicobacter pylori (H. pylori) infection is a major risk factor for chronic gastritis, peptic ulcer disease, and gastric cancer, while the virulence-associated proteins CagA and VacA are closely linked to disease severity. Rapid serological detection that simultaneously identifies infection status and virulence-associated antibody profiles would therefore be valuable for point-of-care risk assessment. In this study, we developed a europium nanoparticle-based multiplex fluorescent lateral flow immunoassay (LFIA) for the simultaneous detection of serum antibodies against Ure, CagA, and VacA. Single-plex assays were first established to verify feasibility, showing good sensitivity, reproducibility, and diagnostic performance, with area under the curve (AUC) values of 0.938, 0.949, and 0.951 for anti-Ure, anti-CagA, and anti-VacA, respectively. These assays were subsequently integrated into a multiplex platform through systematic optimization of test-line arrangement, probe loading, and reaction time. The optimized multiplex LFIA completed detection within 15 min and exhibited high analytical sensitivity, with detectable limits of serum dilution up to 1 : 160, 1 : 320, and 1 : 160 for anti-Ure, anti-CagA, and anti-VacA, respectively. Compared with commercial immunochromatographic kits, the multiplex assay showed improved sensitivity for anti-Ure and anti-CagA, while maintaining comparable performance for anti-VacA. The assay also demonstrated good inter-batch precision and favorable storage stability. Clinical evaluation using 100 serum samples yielded AUC values of 0.932, 0.993, and 0.984 for anti-Ure, anti-CagA, and anti-VacA, respectively, with overall concordance rates of 89.0%, 97.0%, and 95.0%. Collectively, this multiplex fluorescent LFIA provides a rapid, sensitive, and robust point-of-care platform for simultaneous H. pylori serodiagnosis and virulence-associated antibody profiling.