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Updated: May 26, 2025
Optimization of a Quantitative Micro-neutralization Assay
Published on: December 14, 2016
Development and validation of a quantitative Orthopoxvirus immunoassay to evaluate and differentiate serological
Joanne Byrne1, Gurvin Saini2, Alejandro Garcia-Leon2
1Centre for Experimental Pathogen Host Research (CEPHR), University College Dublin, Belfield, Dublin 4, Ireland; Department of Infectious Diseases, St Vincent's University Hospital, Elm Park, Dublin 4, Ireland.
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.
