BACKGROUND: The Mpox outbreak, caused by Monkeypox virus (MPXV), underscores the need for a serological assay to assess Mpox immunity. Modified Vaccinia Ankara (MVA) vaccine, an attenuated vaccinia virus (VACV), is authorised for Mpox prevention. We aimed to develop a quantitative immunoassay to differentiate infection- and vaccination-induced immunity and explore serological responses to Mpox infection and vaccination. METHODS: We evaluated an electrochemiluminescence assay targeting IgG to 10 MPXV and 3 VACV antigens in plasma from adults in a cohort study with previous Mpox, MVA-vaccination, or historical controls. Sensitivity and specificity to distinguish i) seropositive versus naive and ii) infection- versus vaccination-induced seropositivity were determined using ROC curves. Antibody kinetics were analysed with generalised additive models. FINDINGS: Eight of the thirteen IgG antibodies showed significant titre differences across groups identifying three key antigens: MPXVB6R, MPXVA27L, and VACVB5. A VACVB5 IgG titre of 0.082 IgG normalised units (nu) offered 74% (95% CI: 59-82%) sensitivity and 81% (73-96%) specificity for previous antigen exposure (infection or vaccine). For infection alone, an MPXVB6R IgG titre of 0.075 IgGnu provided 89% (82-98%) sensitivity and 94% (86-100%) specificity. To differentiate infection from vaccination-induced seropositivity, the sum of MPXVA27L IgG and the B6R/VACVB5 ratio provided 89% (80-96%) sensitivity and 80% (74-84%) specificity. VACVB5 IgG titres declined over time, with higher titres post-Mpox than post-vaccination (p <
0.0001). INTERPRETATION: This assay demonstrates high sensitivity and specificity in quantifying and differentiating between antibody responses to Mpox infection and vaccination. Post-Mpox antibody responses were higher than post-vaccination, though both waned over time. FUNDING: Health Research Board (MONKEYVAX-2022-1), University College Dublin School of Medicine.