Development of a genus-specific antigen capture ELISA for orthopoxviruses. Target selection and optimized screening

Raw data for quantification of anti surface protein antibody binding to vaccinia virus.

Method description

Immuno-negative staining and electron microscopy were performed as described elsewhere (Laue, 2010). Briefly, purified VACV_NYCBOH particles were inactivated by incubation in freshly prepared 2% PFA in 0.05 M HEPES (pH 7.2), sonicated and immobilized on sample supports for transmission electron microscopy. Biotinylated pAbs were titrated on BSA coated grids, until detection with 5 nm gold nanoparticle coupled streptavidin (British Biocell, Cardiff, United Kingdom) resulted in the same mean background labelling density of ~10 particles per view field at a, 87,000-fold magnification (anti-A27: 0.7 µg/mL; anti-D8: 2 µg/mL; anti-H3: 0.9 µg/mL; anti-L1: 1.9 µg/mL). Negative staining was performed with either 0.1 or 0.5% uranyl acetate solution. For quantification, only IMV particles of the mulberry form, which were found isolated from other particles, were analyzed. Randomized sampling was done in 22 evenly distributed mesh areas with five viral particles analyzed per area. Imaging was done with a Tecnai 12 BioTwin (FEI Corp.) at 120 kV and a 1k digital CCD camera (Megaview III, Olympus Soft Imaging Solutions).