Antibodies and proteins
The diagnostic ABICAP is based on two monoclonal antibodies (mAbs) targeting the OPV surface protein A27: mAb A1/40 and mAb A3/710. Briefly, both antibodies bind to A27 with high affinity (A1/40: 2.3 nM; A3/710: 4.3 nM) and recognize non-overlapping linear epitopes located in close proximity to the N-terminal heparin-binding domain of A27 (A1/40: aa 24–38; A3/710: aa 13–27). Both antibodies have previously been selected in an antigen capture ELISA, representing the best combination to detect native VACV, CMLV, CPXV, ECTV, and MPXV. Production, purification, storage, and biotinylation of antibodies was performed as described previously . The following reagent was obtained through the NIH Biodefense and Emerging Infections Research Resources Repository (BEI Resources), NIAID, NIH: Vaccinia virus (WR) rA27 (NR-2622) with a C-terminal histidine tag, recombinantly expressed from baculovirus.
The virus panel used for development and validation included VACV strain New York City Board of Health (NYCBOH, VR-1536™, ATCC/LGC Standards GmbH, Wesel, Germany) and CPXV strain GuWi . Camelpox virus (CMLV) strain CP-19, Ectromelia virus (ECTV) strain Nü-1, and MPXV strain MSF6 were kindly provided by Prof. Dr. Hermann Meyer (Bundeswehr Institute of Microbiology, Munich, Germany). The OPV ABICAP detection system was additionally validated with two VARV strains, VARV Solaiman 1974 and VARV Niger 1969. Parapoxvirus (PPV) ORF D1701 was kindly provided by Achim Rziha (Friedrich-Loeffler-Institute, Tübingen, Germany) whereas herpes simplex virus 1 (HSV-1) was isolated in our lab from a patient. Propagation and titration of viruses on cell culture was done according to standard procedures . Viruses were used as clarified supernatant (HSV, PPV, MPXV, and VARV) or as semi-purified viral particles by centrifugation through a 40% sucrose cushion . All viruses were used natively except for MPXV which was heat-inactivated at 60 °C for 2 h. All work with live VARV was conducted within a biosafety level 4 laboratory in accordance to guidelines and approvals from the World Health Assembly Advisory Committee on Variola Virus Research.
For final validation, a panel containing inactivated (γ-irradiation) highly pathogenic viruses comprising Yellow fever virus strain 17D, Ebola virus strain Zaire, Marburg virus, VACV, and MPXV in Dulbecco’s Modified Eagle Medium was kindly provided by the P.R.O.B.E. consortium. Viruses in this panel have been quantified by quantitative real-time PCR using published assays [14, 15] and were investigated in a blinded manner to test the suitability of the ABICAP assay to discern highly pathogenic viruses in a bioterrorist setting.
Clinical sample material
Clinical sample material tested in this work had been sent to the Robert Koch Institute for poxvirus diagnostics. Crust material and surface swabs were delivered dry and suspended in 500 μL of phosphate buffered saline (PBS, pH 7.4) without magnesium and calcium. Surface swabs were thoroughly mixed on a vortexer while crusts were homogenized in a Fastprep 24 homogenizer (MP Biomedicals, Eschwege, Germany) after addition of Precellys® ceramic beads (1.4 mm; PEQLAB, Erlangen, Germany), and DNA was prepared from 200 μL of sample using the Qiagen Blood and Tissues Kit (Qiagen, Hilden, Germany) according the manufacturer’s recommendations. Quantitative real-time PCR for OPV diagnostics was performed on 5 μL of isolated DNA, using rpo18 as target as published previously . The number of genome equivalents (GE) contained in the samples was estimated based on the published calibration curve of the rpo18 assay. To test the specificity of the ABICAP assay, clinical samples negative in the OPV PCR were also tested. For differential diagnostics, different PCR assays targeting Molluscipox virus , Parapox virus , Myxoma virus  were tested on isolated DNA from homogenized crusts or swabs. Homogenized samples in PBS were tested in the ABICAP assay after dilution in UCBS casein buffer (SDT, Baesweiler, Germany).
OPV ABICAP assay
The preparation of ABICAP columns was done as described before , using mAb A1/40 immobilized on polyethylene filter frits contained inside the ABICAP columns and biotinylated mAb A3/710 as the detection antibody.
To this aim, polyethylene filter frits (Type 180; Porex, Aachen, Germany) used for the coating of the capture antibodies were activated by successive 10-min washes with 96% ethanol, 50% ethanol, and 3 × coating buffer (0.1 M NaHCO3/ Na2CO3, pH 9.5). All activating and coating steps were done at room temperature under constant stirring and vacuum. The frits were then coated with 7.5 μg/frit of mAb A1/40 in coating buffer (75 μg/mL) overnight and blocked for 25 min with PBS-T BND (PBS + 0.05% Tween-20 + 0.05% BND [5-bromo-5-nitro-1,3-dioxane; SDT, Baesweiler, Germany]) + 0.2% bovine serum albumin (BSA; Carl Roth, Karlsruhe, Germany). Finally, frits were prepared for drying by 1 h of incubation with PBS-T BND + 1% BSA + 5% saccharose and air-dried at 35 °C for 30 min in a fluid bed dryer (FBD2000; Endecotts, London, UK). The columns were equipped with antibody-coated frits between two preblocked separation frits (Type 187; Porex) and stored dry at room temperature until further use.
The principle of ABICAP™ immunofiltration assay (Antibody Immuno Column for Analytical Processes, Senova GmbH, Weimar, Germany) is shown in Fig. 1. 500 μL of pre-diluted sample material (UCBS casein buffer, SDT) were applied per column and incubated for 6 min. After a washing step with 750 μL of washing buffer (PBS-T BND + 0.1% BSA), 500 μL of biotinylated mAb A3/710–20 (in AA1-buffer [SDT]) were added and incubated for 6 min. Columns were washed again and incubated with 500 μL of streptavidin-PolyHRP 40 (SA-pHRP; in SA1-buffer, both SDT) for 6 min. Two washing steps later (1 × 750 μL of washing buffer, 1 × 750 μL of substrate buffer [phosphate/citrate buffer, pH 5.0]) 500 μL of precipitating TMB substrate (epTMB; SDT) were added for 6 min. Finally, columns were washed with 750 μL of substrate buffer, and the extinctions were read at 525 nm with a handheld photometer device (Senova; Fig. 1).
To allow for maximum sensitivity of detection at low background, both SA-pHRP and biotinylated detection antibodies were titrated (0.1, 0.5, 1, and 5 μg/mL) in the absence of antigen. For titration of SA-pHRP, the protocol was performed as described, but instead of biotinylated detection antibody AA1-buffer only was added. For titration of biotinylated detection antibodies, SA-pHRP was added at the optimum concentration determined in the previous experiment. Low background was obtained with mAb A3/710 at 0.5 μg/mL and SA-pHRP at 0.5 μg/mL (Additional file 1: Figure S1).
The cutoff to determine the limits of detection (LOD) and to discern positive from negative samples was calculated as the mean + 3 × standard deviation of negative control columns (buffer only, n = 16). The assay’s sensitivity was determined by measuring the LOD for recombinant A27. The assay’s specificity was determined for various OPV strains by using purified viral particles and by testing clinical crust material from CPXV-infected patients. To determine the test’s specificity, additional viruses causing similar clinical pictures were used, including HSV-1 and PPV. The LOD was determined by measuring serial dilutions of semi-purified virus particles and virus from clarified cell culture supernatant. Finally, the OPV ABICAP assay was validated with a blinded panel of PCR-quantified highly pathogenic viruses, including Yellow Fever virus, Ebola virus, Marburg virus, VACV, and MPXV.