Figure 6.

Nonspecific binding analysis for the AF1–Fc antibody relative to other scFv–Fc and IgG antibodies. The average levels of nonspecific binding (signal/background) for scFv–Fc and IgG antibodies (100 nm) were evaluated using six immobilized molecules (cardiolipin, keyhole limpet hemocyanin, lipopolysaccharide, ssDNA, dsDNA, and insulin). The 4D5, AF1, A10, and B2 antibodies are scFv–Fc antibodies with a common human IgG1 Fc fragment, and they were detected using a goat anti-human Fc antibody. AF1 and AF1* are the same scFv–Fc antibodies, but AF1* was detected using an anti-FLAG antibody instead of an anti-Fc antibody. The human IgGs (mAbs) have human IgG1 or IgG2 Fc fragments, and they were detected using a goat anti-human Fc antibody. The background values were evaluated without primary antibody and with immobilized polyspecificity molecules. The nonspecific binding values (signal/background) for each immobilized molecule were evaluated separately and then averaged together for each antibody. The data are average values for three independent experiments, and the error bars are standard deviations.