Development of a specific MPXV antigen detection immunodiagnostic assay

Abstract

Human monkeypox (mpox) has recently become a global public health emergency; however, assays that detect mpox infection are not widely available, largely due to cross-reactivity within the Orthopoxvirus genus. Immunoassay development was largely confined to researchers who focus on biothreats and endemic areas (Central and West Africa) until the 2022 outbreak. As was noted in the COVID-19 pandemic, antigen detection assays, integrated with molecular assays, are necessary to help curb the spread of disease. Antigen-detecting immunoassays offer the advantage of providing results ranging from within min to h and in lateral flow formats; they can be deployed for point-of-care, home, or field use. This study reports the development of an mpox-specific antigen detection immunoassay developed on a multiplexed, magnetic-bead-based platform utilizing reagents from all research sectors (commercial, academic, and governmental). Two semi-quantitative assays were developed in parallel and standardized with infectious mpox virus (MPXV) cell culture fluid and MPXV-positive non-human primate (NHP) sera samples. These assays could detect viral antigens in serum, were highly specific toward MPXV as compared to other infectious orthopoxviruses (vaccinia virus, cowpox virus, and camelpox virus), and exhibited a correlation with quantitative PCR results from an NHP study. Access to a toolbox of assays for mpox detection will be key for identifying cases and ensuring proper treatment, as MPXV is currently a global traveler.

Keywords: Magpix; antigen assay; clade-specific; immunofluorescence; mpox.

Figure 1

Assay development (A), and false color transmission electron microscope (TEM) image of MPXV in a liver cell (B).

Figure 2

Screening of MAbs and detection strategy with recombinant antigens. The 1° MAbs (vertical axis) are covalently linked to magnetic microspheres. 2° MAbs (horizontal axis) are biotinylated, and detection is achieved with SAPE. Clade I MPXV A29 is shown in (A), and Clade II MPXV A29 is shown in (B). Clade selectivity of down-selected MAb pairs using 2°-biotin conjugate, as shown in (C), and 2°-PE conjugate, as shown in (D). Data are presented as signals using MPXV A29 divided by signals using VACV A27.

Figure 3

Limits of detection with infectious virus. α-A29 is used as the capture MAb, with S27 or 4B4 as the 2° MAb. S27-PE, S27-B, and 4B4-B are depicted as black circles, blue squares, and red triangles, respectively. Median fluorescence intensity (MFI) is shown as a function of virus titer in pfu/mL for MPXV strains Zaire '79 (A), Katakombe (B), US 2003 (C), and the currently circulating strain (D). Data were fit with a 4-parameter sigmoidal function. Interpolated limits of detection of the antigen assays are shown with 90% confidence intervals in (E). The 4B4 assay does not detect Clade II MPXV and is excluded from (C, D).

Figure 4

Detection of MPXV with specific assays. NHPs were challenged on day 0 and treated with ST-246 (hollow) or placebo (filled) at 72 h. Serum was assayed using α-A29 as the 1° MAb, with S27-PE (black circles) or 4B4-B (red triangles) as the 2° MAb. qPCR data are shown as blue diamonds. Error bars are SEM for 3 treated or 4 placebo NHPs.