The Use of Distinctive Monoclonal Antibodies in FMD VLP- and P1-Based Blocking ELISA for the Seromonitoring of Vaccinated Swine.

The serum neutralization (SN) test has been regarded as the "gold standard" for seroconversion following foot-and-mouth disease virus (FMDV) vaccination, although a high-level biosafety laboratory is necessary. ELISA is one alternative, and its format is constantly being improved. For instance, standard polyclonal antisera have been replaced by monoclonal antibodies (MAbs) for catching and detecting antibodies, and inactive viruses have been replaced by virus-like particles (VLPs). To the best of current knowledge, however, no researchers have evaluated the performances of different MAbs as tracers. In previous studies, we successfully identified site 1 and site 2 MAbs Q10E and P11A. In this study, following the established screening platform, the VLPs of putative escape mutants from sites 1 to 5 were expressed and used to demonstrate that S11B is a site 3 MAb. Additionally, the vulnerability of VLPs prompted us to assess another diagnostic antigen: unprocessed polyprotein P1. Therefore, we established and evaluated the performance of blocking ELISA (bELISA) systems based on VLPs and P1, pairing them with Q10E, P11A, S11B, and the non-neutralizing TSG MAb as tracers. The results indicated that the VLP paired with S11B demonstrated the highest correlation with the SN titers (R2 = 0.8071, n = 63). Excluding weakly positive serum samples (SN = 16-32, n = 14), the sensitivity and specificity were 95.65% and 96.15% (kappa = 0.92), respectively. Additionally, the P1 pairing with Q10E also demonstrated a high correlation (R2 = 0.768). We also discovered that these four antibodies had steric effects on one another to varying degrees, despite recognizing distinct antigenic sites. This finding indicated that MAbs as tracers could not accurately detect specific antibodies, possibly because MAbs are bulky compared to a protomeric unit. However, our results still provide convincing support for the application of two pairs of bELISA systems: VLP:S11B-HRP and P1:Q10E-HRP.

Novel application of Influenza A virus-inoculated chorioallantoic membrane to characterize a NP-specific monoclonal antibody for immunohistochemistry assaying.

Monoclonal antibodies (MAbs) are widely applied in disease diagnoses. Herein, we report a MAb, WF-4, against Influenza A virus nucleoprotein (NP), its broad response with Influenza A virus, and its application in an immunohistochemistry (IHC) assay. WF-4 was screened by immunofluorescence assay (IFA). The results showed that its reactivity with baculovirus-expressed full-length recombinant NP (rNP) in Western blot (WB), indicating its IHC applicability. Fifteen Influenza A virus (reference subtypes H1 to H15) infected chicken embryonated chorioallantoic membranes (CAM), fixed by formalin, were all detectable in the WF-4-based IHC assay. Also, the reactivity of the IHC test with NP from experimentally inoculated H6N1 and from all recent outbreaks of H5 subtype avian Influenza A virus (AIV) field cases in Taiwan showed positive results. Our data indicate that CAM, a by-product of Influenza A virus preparation, is helpful for Influenza A virus-specific MAb characterization, and that the WF-4 MAb recognizes conserved and linear epitopes of Influenza A virus NP. Therefore, WF-4 is capable of detecting NP antigens via IHC and may be suitable for developing various tests for diagnosis of Influenza A virus and, especially, AIV infection.

Peptides mimicking viral proteins of porcine circovirus type 2 were profiled by the spectrum of mouse anti-PCV2 antibodies.

BACKGROUND: Porcine circovirus 2 (PCV2) is a small, non-enveloped DNA virus causing swine lymphocyte depletion and severe impact on the swine industry. The aim of this study was to evaluate the antigenicity and immunogenicity of specific peptides, and seeking the potential candidate of PCV2 peptide-based vaccine. It's initiating from peptides reacting with PCV2-infected pig sera and peptide-immunized mouse sera. RESULTS: The data showed that the sera from PCV2-infected pigs could react with the N-terminal (C1), middle region (C2), and C-terminal peptide (C3) of PCV2 capsid protein (CP), ORF3 protein (N1), ORF6 protein (N2) and ORF9 protein (N3). This study demonstrated that anti-PCV2 mouse antisera could be generated by specific synthetic peptides (C3 and N2) and recognized PCV2 viral protein. We found that the tertiary or linear form C-terminal sequence (C3) of PCV2 capsid peptide only appeared a local distribution in the nucleus of PCV2-infected PK cells, virus-like particles of PCV2 major appeared a local distribution in the cytoplasm, and ORF 6 protein of PCV2 were shown unusually in cytoplasm. Furthermore, most residues of the C1 and the C3 were presented on the surface of PCV2 CP, in the view of 3-D structure of the CP. Our data demonstrated that PCV2-infected pigs had higher OD405 value of anti-C3 IgG on Day 1, Month 3 and Month 6 than in Month 1. These pigs had higher anti-C3 IgM level in Month 3 and Month 6 than on Day 1 (P < 0.01). CONCLUSIONS: We demonstrated that the key peptide (C3) mimic the C-terminal of PCV2 capsid protein which were capable of inducing antibodies. The specific antibody against the C3 were confirmed as the serological marker in PCV2-infected pigs.