Figure 2

Engineering StrepTagII as antigen and development of anti-StrepTagII monoclonal antibodies. (a) Schematic representation of the StrepTagII antigen expressed in loop 2 of outer membrane protein X (OmpX) in the outer membrane of E. coli. OmpX structure obtained from the protein data bank⁶⁵. (b) Mass photometry density plot of IgM anti-StrepTagII expressed in the presence of the J-chain. Annotated masses correspond to local maxima in density and the peak indicated in blue represents a common contaminant of about 80–90 kDa, possibly a half IgM monomer or single heavy chain. (c) Binding of a concentration range of anti-StrepTagII IgM, IgG1, and IgG4 to either MG1655-OmpX-StrepTagII bacteria (solid lines) or MG1655 wildtype bacteria (dotted lines). Antibody binding was detected with Goat-anti-human-kappa-AF488. (d) Inner membrane damage (% Sytox positive) of MG1655-OmpX-StrepTagII bacteria treated either with buffer (0%) or with 1.25% serum equivalent complement components with either no antibody (No Ab), 1 µg/mL anti-StrepTagII IgM, anti-StrepTagII IgG1, or anti-StrepTagII IgG4. Samples (c and d) were measured by flow cytometry and data represent mean ± SD of three independent experiments.