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 (ASF) is an acute, febrile, and lethal pig disease induced by the African swine fever virus (ASFV). In the absence of an effective vaccine, early diagnosis is essential for the prevention and control of ASF disease. The p54 protein is important for ASFV diagnosis and vaccine design. In this study, ASFV p54 protein was constructed, expressed, purified, and used to generate three mAbs, namely 9A3, 5H2, and 2G6. Epitope mapping was performed using alanine mutants; the minimal linear epitope recognized by 9A3 and 5H2 was ⁵⁶KKKAAAI⁶², and the minimal linear epitope recognized by 2G6 was ¹⁰⁸TNRPATN¹¹⁴. Of these, ⁵⁶KKKAAAI⁶² was identified as a new linear epitope for the first time. The epitopes were highly conserved in at least genotypes I and II. Alanine-scanning mutagenesis further revealed that residues 56K, 57K, 60A, 61A, 62L, 108T, 110R, 111P, 113T, and 114N were the core sites involved in antibody recognition. Overall, the mAbs and epitopes of the p54 protein identified in this study provide theoretical support for the development of ASFV vaccines based on the B cell epitope, the development of ASFV therapeutic antibody drugs, and the development of ASFV diagnostic tools.

Background: The HPV16 E6 oncoprotein facilitates the ubiquitin-mediated degradation of the tumor suppressor p53, constituting a pivotal mechanism underlying viral immune evasion, cellular immortalization, and ultimately, malignant transformation. This study aimed to develop a reliable detection tool for the HPV16 E6 protein. Methods: Recombinant GST-E6 and E6-His proteins were expressed and purified using a prokaryotic expression system. Female BALB/c mice were immunized with GST-E6, and two hybridoma cell lines (G11A11 and A4) stably secreting anti-HPV16 E6 monoclonal antibodies were generated via hybridoma technology. Antibody pairing experiments identified A4 and G11A11 as suitable for sandwich ELISA. The optimal detection system was established using A4 antibody at 2 μg/mL for coating, HPV16 E6-His at 5 μg/mL as the detection antigen, and G11A11-HRP at a 1:200 dilution as the detection antibody. To validate the reliability, Hacat-HPV16E6 cell lysates were tested in parallel with a commercial ELISA kit. Results: After purification, the titers of both antibodies reached 1:204,800. The lower limit of quantification (LOQ) was 4.79 ng/mL and the limit of detection (LOD) was 3.39 ng/mL. The comparison with the commercial kit showed good consistency, with percentage differences ranging from 20% to 40%, confirming that the established ELISA is reliable for quantitative detection. Conclusions: This study successfully yielded high-titer and highly specific anti-HPV16 E6 monoclonal antibodies and developed a specific double-antibody sandwich ELISA, thereby furnishing a technical foundation for both fundamental research and laboratory-based applications related to HPV16-associated tumors.