A chemiluminescence immunoassay for detecting neutralizing antibodies of foot-and-mouth disease virus serotype A

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Foot-and-mouth disease (FMD), caused by the foot-and-mouth disease virus (FMDV), is an extremely infectious disease that affects animals possessing cloven hooves. The disease spreads rapidly over long distances, with over 70 susceptible species, posing severe threats to global livestock industries (Kitching et al. 2007 ; Arzt et al. 2024 ). FMDV, a small RNA virus with high genetic variability, is classified into seven serotypes: O, A, C, Asia 1, SAT 1, SAT 2, and SAT 3 (Bachrach 1968 ). There is a lack of cross-protection between different serotypes of the virus. Moreover, even within the same serotype, antigenic disparities among various lineages restrict the cross-protection against heterologous strains (Belsham 2005 ). In China, FMDV serotype A and serotype O are predominant. FMDV serotype A demonstrates the most significant antigenic structural variability, resulting in limited cross-protection among its different lineages (Fry et al. 1999 ; Kitching et al. 2007 ; Islam et al. 2021 ). In regions where FMD is endemic, inactivated vaccines continue to be the principal preventive approach (Lu et al. 2022 ). As the principal protective immune components mediating host defense against FMDV infection, neutralizing antibodies serve as critical correlates for post-vaccination immune monitoring. Quantitative assessment of these antibodies provides an evidence-based foundation for evaluating vaccine effectiveness and optimizing immunization protocols (Amadori et al. 1991 ; Pay and Hingley 1992 ). The World Organization for Animal Health (WOAH) recommended three serological assays: virus neutralization test (VNT), liquid-phase blocking enzyme-linked immunosorbent assay (LPB-ELISA) and solid-phase competitive enzyme-linked immunosorbent assay (SPC-ELISA) (WOAH 2024 ). VNT is the “gold standard” for detecting neutralizing antibodies; however, this method requires using live viruses, posing a risk of virus dissemination. Moreover, it has a long experimental cycle and poor repeatability and is not suitable for batch testing of serum samples (Jamal and Belsham 2013 ). LPB-ELISA is well suited for large-scale detection, but it has a propensity for non-specific reactions, which frequently leads to false positive results (Hamblin et al. 1986 ). SPC-ELISA exhibits operational simplicity and robust reproducibility; however, its low-throughput design significantly hinders efficient processing of large sample volumes (Mackay et al. 1998 ; Brocchi et al. 2006 ). Therefore, there is a need to develop a safe, precise, and rapid novel method for the detection of neutralizing antibodies.

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