R409K mutation prevents acid-induced aggregation of human IgG4
Fig 5
Effect of R409K mutation on antigen (FcγR) binding and CDC activity.
(A) Binding of IgG4 mutants to Raji cells, measured using flow cytometry; cells were stained with IgG4 (open circles), IgG4PE (closed squares), IgG4PE_R409K (open squares), or isotype control IgG (closed circles), and the binding was detected using an Alexa Fluor 488-labeled secondary antibody. (B) ELISA for measuring the binding activity of IgG4 mutants toward human FcRs. Each FcγR was coated on 96-well immunoplates by using anti-tetra-His antibodies, and the plates were incubated with the indicated concentrations of IgG1 (closed circles), IgG4 (open circles), IgG4PE (closed squares), or IgG4PE_R409K (open squares); binding was detected using a peroxidase-labeled secondary antibody. (C) CDC of IgG4 mutants against Raji cells. After 2-h incubation, cytotoxicity was measured using CellTiter-Glo. Means ± SD of triplicates are shown.