Quantifying TRIM21-Antibody Interactions

Recombinant AdV5 hexon (AbD Serotec) (diluted to 1 µg/ml in PBS), a polyclonal anti-human Fc Ab produced in goat (locally produced; diluted to 1 µg/ml in PBS), or AdV5-GFP (Sino Biologicals) (diluted 1:1000 in PBS from a 1.0 × 10¹⁰ PFU/ml viral stock) was coated in 96-well plates (Nunc) and incubated overnight at 4°C. Remaining surface area was blocked using PBS/4% skim milk (S) (Acumedia), before washing four times with PBS/0.005% Tween 20 (T). Titrated amounts of h9C12 variants diluted in PBS/T/S were added to the wells and incubated for 1 h at room temperature (RT). Following washing as above, MBP-tagged full-length human TRIM21 or an alkaline phosphatase (ALP)-conjugated polyclonal anti-human Fc Ab from goat (Sigma-Aldrich), each diluted to 1 µg/ml in PBS/S/T, was added and incubated for 1 h at RT. Following washing as above, bound MBP-tagged full-length human TRIM21 was detected by an HRP-conjugated anti-MBP Ab (New England Biolabs). Binding was visualized by addition of tetramethylbenzidine solution (Calbiochem) or ALP substrate (Sigma-Aldrich). The HRP-tetramethylbenzidine reaction was terminated by addition of 100 µl 1 M HCl, and the 450-nm (HRP) or 405-nm (ALP) absorption spectrum was recorded using a Sunrise plate reader (TECAN).