Development of an On-Site Rapid Detection Method for Porcine Pseudorabies Virus Using gD Protein-Based Monoclonal Antibody and Red Latex Microsphere Immunochromatographic Strip

Pseudorabies, an acute multi-host zoonotic infectious disease, is etiologically caused by the pseudorabies virus (PRV). As a naturally occurring infection in swine, pseudorabies imposes substantial economic losses on the global swine industry. Furthermore, the emergence of cross-species human infections caused by PRV since 2017 highlights its previously underestimated zoonotic threat and public health significance. The objective of the present study was to establish an innovative double-antibody sandwich lateral flow immunoassay (DAS-LFIA) for the rapid detection of PRV gD protein. Initially, recombinant PRV gD protein was generated for murine immunization, followed by the screening and isolation of PRV-specific monoclonal antibodies (mAbs). For DAS-LFIA establishment, paired mAbs were selected and optimized. The mAb-9B11 conjugated with red latex microspheres acts as the detection probe. Simultaneously, PRV-gD-targeting mAb-2F6A11 and Staphylococcal protein A (SPA) were separately immobilized onto the nitrocellulose membrane to serve as the test (T) line and control (C) line, respectively. The as-developed DAS-LFIA demonstrated a high degree of specificity, with no detectable cross-reactivity against common swine pathogens or other PRV-encoded glycoproteins, namely gB, gC, and gE. Furthermore, the DAS-LFIA exhibited high analytical sensitivity, achieving a visual limit of detection (vLOD) of 0.31 ng/μL for gD protein, with visual readout obtainable within 5 min. Owing to its operational simplicity, rapid turnaround, and analytical sensitivity, the DAS-LFIA constitutes a practical and robust tool for point-of-need PRV surveillance, particularly in resource-constrained environments, with substantial value for curating PRV transmission within the swine industry and mitigating associated public health risks.