Fig. 3. Binding competition experiments between different pairs of antibodies interacting with the Prn antigen. (A and C) MS1 spectra obtained from incubation of 2B1 (5 µM) with either 1F2 (5 µM) or 1E7 (5 µM) and Prn (5 µM), with deconvoluted spectra shown in the insets on the right. (B and D) Schematic representations of Prn + 2B1-Fab + 1F2-Fab and Prn + 2B1-Fab + 1E7-Fab, respectively, with Prn shaded gray (1DAB), and AlphaFold-predicted structures for 2B1-Fab shaded pink, 1F2-Fab shaded blue and 1E7-Fab shaded purple, all shown as molecular surfaces. The model in 3D displays the potential overlap of 2B1 and 1E7 that would impede simultaneous binding. The presence of high intensity ion peaks centered at m/z 9000 corresponds to complexes containing Prn and two different antibodies that bind to distant epitopes such as 2B1 and 1F2 and demonstrates that two antibodies can simultaneously bind to the Prn antigen. Conversely, simultaneous binding of antibodies that bind to overlapping or proximate epitopes (2B1 and 1E7) is suppressed, resulting in low intensity peaks for the [Ab]₂·[Prn]₂ complexes.