| Summary: | <b>Background</b>: Despite its global eradication in 1977, smallpox remains a concern owing to its potential as a biological agent, thereby prompting the ongoing development and utilization of its vaccine. Vaccination with the <i>Vaccinia</i> virus induces immunity against variola virus, the causative agent of smallpox; however, this immunity does not extend to viruses of different genera within the <i>Poxviridae</i> family. In this study, we aimed to assess the efficacy of an enzyme-linked immunosorbent assay (ELISA) method utilizing <i>Vaccinia</i> virus and recombinant A27L antigen for detecting antibodies against smallpox. <b>Methods.</b> An analysis of the serum from 20 individuals pre- and post-vaccination with the CJ strain (CJ50300) revealed neutralizing antibodies, which were confirmed using the plaque reduction neutralization test (PRNT). The ELISA method, validated with a PRNT<sub>50</sub> cut-off value of >4, exhibited a sensitivity and specificity of >95% and was particularly reactive with the inactivated virus. Furthermore, adherence to the smallpox vaccination policy revealed significant differences in <i>Orthopoxvirus</i> antibody levels among 300 individuals of different age groups. These findings highlight the reliability and efficacy of the ELISA method in detecting post-vaccination antibodies and contribute significantly to diagnostic methods to prepare for potential smallpox resurgence and bioterrorism threats.
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