An improved and high-throughput mpox virus microneutralization assay

Since the 2022 global mpox outbreak, the lack of specific antibodies for mpox virus (MPXV) detection has hindered precise immunoassays due to cross-reactivity among Orthopoxviruses (OPXVs). This study developed and characterized two monoclonal antibodies (mAbs), CML01 and CML02, targeting the MPXV A35 protein, a conserved surface antigen of extracellular virions. Cross-reactivity assessments via enzyme-linked immunosorbent assay (ELISA), western blot, and indirect immunofluorescence assays (IFA) confirmed that both mAbs bound exclusively to MPXV A35, showing no reactivity with homologous proteins from cowpox (A34), vaccinia (A33), or variola viruses (A36) despite 92.3 %–96.1 % sequence homology. IFA showed recognition of MPXV-infected cells with half-maximal effective concentrations (EC50) of 0.15 and 0.17 μg/mL, respectively. Notably, an IFA-based microneutralization assay using the mAb CML02 exhibited strong correlation (r = 0.93, P < 0.0001) with the traditional plaque reduction neutralization test (PRNT) while enabling higher throughput. Plasma from convalescent mpox patients validated the assay’s utility in testing neutralizing antibody titers. These mAbs address critical gaps in MPXV-specific immunological testing by virtue of their high specificity, which prevents cross-reactivity with other OPXVs and eliminates interference from immunity induced by smallpox vaccination. This work underscores A35 as a key epitope for MPXV-specific immunity and provides essential tools for combating the ongoing mpox threat.