Development and Evaluation of a Multiplex Lateral Flow Immunoassay for Multiple Respiratory Virus Detection

Background: Simultaneous detection of influenza A virus (IAV), influenza B virus (IBV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for rapid differential diagnosis in febrile patients, particularly in the context of co-circulating respiratory viruses. However, conventional gold nanoparticle-based lateral flow immunoassays are limited by single-color readouts and insufficient sensitivity, restricting their multiplex detection capability. This study aimed to develop a multicolor latex-based immunochromatographic strip as a rapid, sensitive, and reliable tool for analyte detection.Methods: In this study, a multicolor latex microsphere-based immunochromatographic assay was developed for the rapid and simultaneous detection of SARS-CoV-2, IAV, and IBV antigens. Colored latex microspheres conjugated with virus-specific antibodies enabled visual differentiation of targets (SARS-CoV-2: red; IAV: blue; IBV: black). The analytical and clinical performance of the assay were systematically evaluated.Results: The assay enabled detection within 15 minutes, with a limit of detection (LOD) of 102 TCID50/mL for all three viruses. Clinical evaluation showed a sensitivity of 97.73% (215/220) and specificity of 100% (200/200) for SARS-CoV-2 detection, with an overall concordance rate of 98.81% (415/420) and a Kappa value of 0.9762 (p < 0.05), indicating excellent agreement with reference methods. Detection consistency for IAV and IBV was 100%. No cross-reactivity was observed with five common respiratory pathogens.Conclusions: This multicolor latex-based immunochromatographic strip exhibits rapid, sensitive, and reliable performance for analyte detection without requiring specialized training. It shows strong potential for home self-testing and early screening in settings where SARS-CoV-2 and influenza co-circulate.