An improved and high-throughput monkeypox virus microneutralization assay

• Scientific question. • The detection of specific antibodies for mpox virus (MPXV) has posed significant challenges owing to cross-reactivity among Orthopoxviruses . • Evidence before this study. • MPXV shares > 90% nucleotide sequence homology with cowpox virus (CPXV), variola virus (VARV), and vaccinia virus (VACV), leading to significant antigenic cross-reactivity. Current MPXV detection and neutralization assays mainly rely on polyclonal antibodies raised against VACV, which lack specificity for MPXV, thereby complicating the differentiation between MPXV infection and infections caused by other OPXVs or immunity induced by smallpox vaccination. Conventional plaque reduction neutralization test (PRNT), while widely used for neutralizing antibody (NAb) against MPXV, suffer from low throughput and technical complexity. • New findings. • The study successfully developed and characterized two monoclonal antibodies (mAbs), CML01 and CML02, which specifically target the MPXV A35 protein. Despite high sequence homology, these mAbs exhibited high specificity with no cross-reactivity with homologous proteins from CPXV, VARV, and VACV. A microneutralization assay based on indirect immunofluorescence assay (IFA) utilizing mAb CML02 showed a strong correlation ( r = 0.93, P < 0.0001 ) with traditional PRNT, thereby enabling higher-throughput detection of neutralizing antibodies against MPXV with specificity. • Significance of the study. • The MPXV-specific monoclonal antibodies eliminate cross-reactivity with other orthopoxviruses, thereby enabling precise differentiation of MPXV infection from immune responses elicited by other OPXVs or prior smallpox vaccination. The development of high-throughput neutralization assay based on MPXV-specific mAb streamlines NAb detection, facilitating rapid evaluation of vaccine efficacy and population immunity during outbreaks. 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 (EC 50 ) 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 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.