Development and Validation of a Competitive ELISA Based on Bovine Monoclonal Antibodies for the Detection of Neutralizing Antibodies against Foot-and-Mouth Disease Virus Serotype A.

The level of neutralizing antibodies in vaccinated animals is directly related to their level of protection against a virus challenge. The virus neutralization test (VNT) is a "gold standard" method for detecting neutralizing antibodies against foot-and-mouth disease virus (FMDV). However, VNT requires high-containment facilities that can handle live viruses and is not suitable for large-scale serological surveillance. In this study, a bovine broadly neutralizing monoclonal antibody (W145) against FMDV serotype A was successfully produced using fluorescence-based single-B-cell antibody technology. Using biotinylated W145 as a detector antibody and another bovine cross-reactive monoclonal antibody, E32, which was produced previously as a capture antibody, a competitive enzyme-linked immunosorbent assay for the detection of neutralizing antibodies (NAC-ELISA) against FMDV serotype A was developed. The specificity and sensitivity of the assay were evaluated to be 99.04% and 100%, respectively. A statistically significant correlation (r = 0.9334, P < 0.0001) was observed between the NAC-ELISA titers and the VNT titers, suggesting that the NAC-ELISA could detect neutralizing antibodies against FMDV serotype A and could be used to evaluate protective immunity.

Selection of Vaccine Candidate for Foot-and-Mouth Disease Virus Serotype O Using a Blocking Enzyme-Linked Immunosorbent Assay.

Foot-and-mouth disease (FMD) is a highly contagious disease and one of the most economically important diseases of livestock. Vaccination is an important measure to control FMD and selection of appropriate vaccine strains is crucial. The objective of this study was to select a vaccine candidate and to evaluate the potential of a blocking ELISA for detecting neutralizing antibodies (NA-ELISA) in vaccine strain selection. Binary ethylenimine inactivated vaccines, prepared from four representative circulating strains (FMDV O/Mya/98, SCGH/CHA/2016, O/Tibet/99, and O/XJ/CHA/2017) belonging to four lineages within three different topotypes of FMD virus (FMDV) serotype O in China, were used to vaccinate cattle (12-13 animals for each strain), sheep (12-13 animals for each strain), and pigs (10 animals for each strain). The results of immunogenicity comparison showed that O/XJ/CHA/2017 exhibited the highest immunogenicity among the four strains in pigs, cattle, and sheep both by NA-ELISA and virus neutralizing test (VNT). Cross-neutralization analysis indicated that O/XJ/CHA/2017 displayed broad antigen spectrum and was antigenically matched with other three representative strains both by NA-ELISA and VNT. In addition, A significant correlation (p < 0.0001) was observed between the NA-ELISA titers and the VNT titers for four representative strains. The results showed that O/XJ/CHA/2017 was a promising vaccine strain candidate and NA-ELISA was comparable to VNT in neutralizing antibodies detection and could be used as the reference test system for vaccine strain selection.

Implication of Broadly Neutralizing Bovine Monoclonal Antibodies in the Development of an Enzyme-Linked Immunosorbent Assay for Detecting Neutralizing Antibodies against Foot-and-Mouth Disease Virus Serotype O.

Vaccination with inactivated vaccines is still the main measure to control foot-and-mouth disease (FMD) in areas where the disease is endemic, and the level of neutralizing antibody in vaccinated animals is directly related to their protection against virus challenge. Currently, neutralizing antibody is mainly detected using the virus neutralization test (VNT) based on cell culture, which is laborious and time-consuming and requires restrictive biocontainment facilities. In this study, two broadly neutralizing antibodies (bnAbs), E46 and F128, were successfully produced using techniques for the isolation of single B cells from peripheral blood mononuclear cells (PBMCs) from bovines sequentially immunized with three topotypes of foot-and-mouth disease virus (FMDV) serotype O. Based on these bnAbs, a blocking enzyme-linked immunosorbent assay (ELISA) for detecting neutralizing antibodies (NA-ELISA) against FMDV serotype O was developed. The specificity and sensitivity of the test were estimated to be 99.21% and 100%, respectively. A significant correlation (P < 0.01) was observed between the NA-ELISA titers and the VNT titers for all sera from vaccinated animals and for all tested strains, suggesting that the NA-ELISA could detect neutralizing antibodies against FMDV serotype O strains of wide antigenic and molecular diversity and could be used for the evaluation of protective immunity.

Indirect ELISA using a multi-epitope recombinant protein to detect antibodies against foot-and-mouth disease virus serotype O in pigs.

Foot-and-mouth disease (FMD) is a devastating animal disease. A previously developed multi-epitope protein B4 vaccine of the FMD virus (FMDV) serotype O provides safety advantages over inactivated vaccines and could be used to prevent and control FMD in pigs. Commercial enzyme-linked immunosorbent assay (ELISA) kits for assessing vaccine efficacy are available for the inactivated vaccines but not for the multi-epitope protein vaccine. In this study, multi-epitope protein B4 was expressed in Escherichia coli, and an indirect ELISA (I-ELISA) was developed to detect antibodies against FMDV serotype O in pigs. The specificity and sensitivity were 96.7% and 95.9%, respectively. B4-vaccinated pigs yielded B4 I-ELISA serum values that were positively correlated with clinical protection against challenge with FMDV serotype O. The I-ELISA's ability to detect antibodies from animals vaccinated with the inactivated vaccine was also evaluated, and the B4 I-ELISA values were significantly positively correlated with liquid-phase blocking ELISA (LPBE) titers (r = 0.6708, p < 0.0001); thus, the I-ELISA was also suitable for detection of antibodies from swine vaccinated with the inactivated vaccine.