Antibody Binding to Immobilized Peptides

Specific binding of the antibody anti-M7 to the immobilized peptides M7 (EQLKKSKTL), or the control peptides scrambled sM7 (KLSLEKQTK), or the peptide M8 (EEFRIHFT)^{41 (link)} was tested in a solid-phase binding with immobilized peptides in 96-well ELISA plates (Nunc). Binding of anti-M7 was detected by addition of biotinylated anti-mouse IgG and subsequent color reaction after the incubation with HRP-coupled streptavidin and TMB-substrate. Specific binding was calculated by subtracting the binding of mouse IgG to the peptides. Alternatively, binding of anti-M7 was tested in western blot on cell lysates from native CHO cells or CHO cells stably transfected with human Mac-1. Full western blots are shown in Supplementary Figure 1. To test binding of the antibody anti-M7 to human leukocytes, anti-M7 was labeled with Alexa Fluor-647 according to the manufacturer’s protocols (Monoclonal Antibody Labeling Kit, Life Technologies). Human leukocytes were isolated from healthy donors by centrifugation and Red Blood Cell lysis, and stimulated with PMA (200ng/ml), incubated with anti-M7-Alexa 647 (1 μg and 5 μg) and antibody binding was quantified by flow cytometry.