To confirm the binding affinity-mode relationship, free D-mannose (Man) was added to compete for pre-formed QR-DiMan•DC-SIGN/R complexes prepared under a PQR of 4:1. DLS analysis showed that the amount of clustered QR-DiMan•DC-SIGNR species was decreased even with addition of just 0.1 mM of Man (Figure ; Figure ). This is a clear indication that the crosslinked clusters correspond to the weaker binding mode, which is more easily displaced than the stronger simultaneous binding. The average 𝐷 h of the smaller species was also reduced to ~30 nm with ≥ 4 mM Man (Figure ), along with the complete disappearance of the clustered species, indicating the eventual breakdown of both binding modes. Moreover, addition of Man to QR-DiMan•DC-SIGN/R complexes also led to a significant, dose-dependent reduction in FRET ratio (Figure ), indicated by the simultaneous reduction of dye fluorescence and recovery of QR fluorescence. These results are fully consistent with free Man induced lectin•glycan-QR complex dissociation. The FRET ratio change with Man C ([Man]) was then fitted with an offset Hill equation to obtain the apparent-inhibition constant (𝐾 i ), which represents the "apparent" [Man] required to inhibit binding by 50% (eq 2; where 𝐹 is the FRET ratio at a particular [𝑀𝑎𝑛], and 𝑛 represents the Hill coefficient; Figure ).