Epitope binning and affinity analysis of LPs 1 to 21 nanobody clones with regard to human APC via BLI. (A) In a BLI epitope binning experiment, the binding of human APC to nanobody was detected based on the spectral shift of the reflected light at the sensor surface loaded with LPs ranging from 1 to 21. The biosensor tips with nanobody-APC complexes were then dipped into TPP-4885 Fab solution. Based on spectral shifts, 8 clones were found to not block TPP-4885 Fab binding to APC, whereas 13 clones were found to compete with TPP-4885 Fab for the same epitope bin and blocked it from binding to APC. (B) Nanobodies that did not block TPP-4885 Fab binding to human APC were selected for an additional binning experiment by BLI. LP7, LP9, LP12, and LP18 were found to not compete with LP2, LP3, and LP10. Values in the table represent binding responses over background (red, competition between analyte and ligand for APC; orange, competition but with measurable analyte binding; and green, no competition). (C) Clustering analysis showed 2 clusters of nanobodies at an approximately unbiased (AU) P value cutoff of 95% (red, AU P values; green, bootstrap probability [bp]; and purple, edge number). (D) Summary of clusters and median length of CDR3. (E) K_D measurements of LPs ranging from 1 to 21 via BLI. Each nanobody was tested against 5 different concentrations of APC. R² of these binding kinetic measurements ranged from 0.92 to 0.99.