Plasmid DNA was used to transform E. coli DH10B for expression of GST fusion proteins. The respective fusion proteins were induced and the soluble form of the protein was purified from E. coli lysates according to the manufacturer's instructions. To prepare an affinity matrix, 2.6 mg of purified AD-4-GST fusion protein was dialysed against coupling buffer and conjugated to AminoLink Plus Coupling Resin (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Four ml of a HCMV hyperimmune globuline preparation, diluted 1:3 (v/v) with PBS, was passed over 2 ml antigen-coupled beads, followed by extensive washing with PBS. Bound IgG was eluted with 0.2 M Glycin-HCl, pH 3.0, in 1 ml fractions and fractions were dialysed against PBS. Total IgG concentration was determined by an ELISA. In brief, polystyrene 96-well plates were coated with 100 ng AffiniPure goat anti-human IgG, Fcc-specific (Jackson Immuno Research, USA) in 0.5 M carbonate buffer, pH 9.6, overnight at 4uC. Serial log 2 dilutions of the eluted fractions in a volume of 50 ml were added and bound IgG was detected by using a polyclonal peroxidase-conjugated goat F(ab) 2 -fragment antihuman IgG, Fcc-specific (Jackson Immuno Research, USA). A human IgG preparation (Jackson Immuno Research, USA) with known concentration was used as standard.