The procedure for blocking infection with H319 antibodies in polarized Vero E6 cells, grown in transwell inserts, was erroneously omitted on page 1104, Section 3.13. (entitled ‘Infectivity Assays’) of [1]. It is important to note that robust blocking with H319 anti-DAF anti-bodies, as shown in Figure 5B, was measured in polarized cells, seeded on filter supports in transwell plates, as described below.
For effective blocking of hantavirus infection with H319 antibodies, Vero E6 cells were seeded (1.5 × 105 cells/well) in 6-mm polyester 0.4 micron transwell inserts (Corning Life Sciences, Tewksbury, MA) and allowed to grow to confluence and polarize for 3–4 days. Formation of tight junctions was evaluated by measuring an increase in transmonolayer electrical resistance (TER) across the cell monolayer, using a voltohmmeter coupled to an Endohm sensor chamber (World Precision Instruments Inc., Sarasota, FL, USA) [2]. Cells were then infected by adding, SNV or HTN inocula (moi = 0.1) to the apical chamber of the transwell plate. To block binding to DAF expressed on the apical cell surface, cells were pretreated with 2.6 µM H319 anti-DAF antibodies for an hour at 37 °C before adding virus.
The figure caption in Figure 5B should now read: Western blotting of polarized, Vero E6 cells infected with HTN and SNV (control), and cells pretreated with DAF blocking antibody (2.6 µM H319).
References and Notes
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