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.

In recent years, food safety concerns associated with veterinary drug residues have aroused significant public attention, highlighting the critical need for continuous monitoring in both animal-derived foods and the environment to safeguard public health. Medroxyprogesterone acetate (MPA), a major anabolic steroid widely used in animal husbandry, poses potential human health risks according to recent studies. Although conventional analytical techniques based on chromatography and immunological assays are effective, they often suffer from complex operations, the need for skilled personnel, high cost, and time consumption. To overcome these limitations, electrochemical immunosensors modified with advanced nanomaterials offer a promising alternative. Herein, a highly sensitive electrochemical immunosensor was developed for trace MPA detection utilizing core–shell SiO₂-PEI@Ag nanoparticles as the sensing matrix. In this nanocomposite, the structural synergy between the SiO₂ core and outer Ag NPs shell not only provides a high-density platform for specific antibody immobilization, but also drastically expands the electroactive surface area and accelerates electron transfer to maximize signal amplification. Benefiting from these synergistic advantages, the constructed immunosensor exhibited a limit of detection (LOD) of 0.014 ng/mL and a wide linear range of 0.03–100 ng/mL. Furthermore, the recovery rates in pork and milk samples were 91.32–97.29% and 95.14–102.54%, respectively. In addition, negligible cross-reaction was observed toward structurally related steroid hormones related to MPA and common antibiotics. The immunosensor shows good stability, reproducibility, and accuracy, demonstrating great potential as a promising candidate to complement conventional tools for future MPA residue screening.

African swine fever virus (ASFV) is a highly pathogenic DNA virus that continues to circulate globally and poses a serious threat to the swine industry. However, the antigenic complexity of ASFV poses formidable challenges for vaccine and diagnostic development. The pE146L protein, which serves as a structural component of the inner envelope, is vitally required for efficient ASFV replication. In this study, the pE146L protein was expressed in E. coli Rosetta (DE3) cells, and five monoclonal antibodies (mAbs) were generated by immunizing BALB/c mice with the purified pE146L. Epitope fine mapping using a panel of truncated overlapping fragments identified five B-cell linear epitopes: aa37–45, aa65–80, aa86–94, aa86–101, and aa115–124, recognized by mAbs 8F3, 1B8, 5C10, 10F4, and 4B7, respectively. Furthermore, synthesized peptides SP1–SP5 were designed based on these sequences, wherein SP5 was prepared as an extended peptide spanning aa110–124 to potentially enhance antibody binding or peptide presentation. These synthetic peptides reacted not only with their respective mAbs but also with ASFV-positive pig sera, among which SP3 exhibited the strongest immunoreactivity. Sequence alignment revealed that all five epitopes were highly conserved among ASFV genotypes I, II, and the emerging I/II recombinant strains. Collectively, these findings identify novel antigenic epitopes of pE146L, providing preliminary candidates that may contribute to the future development of ASFV serological diagnostics.

African swine fever in both domestic and wild pig populations is caused by the extremely infectious African swine fever virus (ASFV). It seriously endangers biodiversity and results in large financial losses for the worldwide pork sector. The major capsid protein p72 is molecularly chaperoned by the ASFV pB602L protein, which is essential to viral assembly. Furthermore, as a nonstructural protein expressed at late stages of infection, pB602L induces a distinct antibody response that may complement existing serological assays based on structural proteins. Given its strong immunogenicity, pB602L represents a promising antigen for developing supplementary diagnostic tools for African swine fever (ASF). In this study, we successfully generated and separated the ASFV pB602L protein, and we verified its responsiveness using serum from pigs infected with ASFV. Additionally, we produced four monoclonal antibody-specific hybridoma cell lines that targeted the pB602L protein exclusively. These cell lines demonstrated high immunoreactivity and responsiveness toward ASFV pB602L. These results highlight the potential enhancement of diagnostic skills. We have detected two previously unknown linear B-cell epitopes (¹³⁸TIDSFL¹⁴³ and ¹⁶⁴TNVDTC¹⁶⁹) using overlapping peptide and truncated protein fragment analysis. Due to their high degree of conservation across various ASFV strains, these epitopes offer trustworthy candidates for the creation of particular diagnostic instruments. This study expands the known ASFV antigenic repertoire by systematically mapping immunodominant epitopes of pB602L. The identified epitopes provide potential molecular targets for the rational design of multi-epitope subunit vaccines.