Rapid and sensitive point-of-care detection of Orthopoxviruses by ABICAP immunofiltration

Abstract

Background: The rapid and reliable detection of infectious agents is one of the most challenging tasks in scenarios lacking well-equipped laboratory infrastructure, like diagnostics in rural areas of developing countries. Commercially available point-of-care diagnostic tests for emerging and rare diseases are particularly scarce.

Results: In this work we present a point-of-care test for the detection of Orthopoxviruses (OPV). The OPV ABICAP assay detects down to 1 × 10⁴ plaque forming units/mL of OPV particles within 45 min. It can be applied to clinical material like skin crusts and detects all zoonotic OPV infecting humans, including Vaccinia, Cowpox, Monkeypox, and most importantly Variola virus.

Conclusions: Given the high sensitivity and the ease of handling, the novel assay could be highly useful for on-site diagnostics of suspected Monkeypox virus infections in areas lacking proper laboratory infrastructure as well as rapid on-site testing of suspected bioterrorism samples.

Keywords: ABICAP; Cowpox virus; Monkeypox virus; Orthopoxvirus; Point-of-care; Rapid detection; Vaccinia virus; Variola virus; Zoonosis; bioterrorism.

Fig. 1

a ABICAP column with antibody-coated frit and handheld photometer device for readout at 525 nm (Senova, Weimar, Germany). b Schematic representation of the ABICAP assay procedure. Plastic columns with capture antibody-coated frits are filled with sample to capture the viral particles with the following steps: (1) Addition of pre-diluted sample material (sample dilution buffer), (2) washing with washing buffer, (3) addition of biotinylated detection antibody, (4) washing with washing buffer, (5) addition of streptavidin-(SA) PolyHRP, (6) two successive washing steps with washing and substrate buffer, (7) addition of precipitating TMB substrate, (8) final washing with substrate buffer, readout with a handheld photometer device

Fig. 2

Titration of recombinant A27 proteins and different OPV strains to determine detection limit for the ABICAP assay. a Results of titration for recombinant A27 (BEI Resources). b Results of titration of Vaccinia virus (VACV), Cowpox virus (CPXV), Camelpox virus (CMLV), Monkeypox virus (MPXV), Ectromelia virus (ECTV), and Variola virus (VARV) on ABICAP columns