Extended Data Fig. 2: Requirements for capture/detection of secreted proteins and characterization of anti-light chain VHHs for antibody capture.
(a) Capture matrix requirements for secreted proteins: epitopes of capture reagent (for example, VHH), detection reagent (for example, fluorescently labeled anti-IgG antibody) and antigen should not overlap. VHHs bind to the light-chain constant region, enabling simultaneous binding to the antigen binding site of VH/VL. To avoid interfering with antibody capture, the detection antibody should bind to the constant region of the heavy chain, preferably the Fc part (CH2/CH3). Binding affinities should be in the low nM to pM range. (b) Determination of binding affinity of anti-mouse κ VHH against recombinant mouse IgG1κ antibody by biolayer interferometry with antibody as ligand and VHH as analyte: KD = 0.0167 ± 0.001 nM, χ2 = 0.1417, R2 = 0.9991. Tested VHH concentrations: 0.16–4.4 nM. (c) Determination of binding affinity of anti-human λ VHH against recombinant human IgG1λ antibody by biolayer interferometry with antibody as ligand and VHH as analyte: KD = 1.35 ± 0.004 nM, χ2 = 0.7345, R2 = 0.9967. Tested VHH concentrations: 0.49–40 nM. (d) Association rates (kon), dissociation rates (koff) and affinities (KD) of VHHs showcasing the benefit of multivalent binding. Values for the anti-human κ VHH have been determined previously by biolayer interferometry for the monovalent (VHH as ligand and Fab as analyte) and bivalent (VHH as ligand and IgG as analyte) interaction and are denoted by an asterisk (*)82. (e) Biolayer interferometry assay to determine epitope overlap of capture and detection reagents. Binding of anti-mouse κ VHH, anti-mouse IgG1 and RBD antigen to a recombinant mouse anti-RBD antibody. Data of VHH binding followed by RBD binding is shown in blue, while the reverse order of binding is shown in purple. (f) Biolayer interferometry assay to determine epitope overlap of capture and detection reagents. Binding of anti-human κ or λ VHHs, anti-human IgG Fc and RBD antigen to recombinant human anti-RBD antibodies. Data of VHH binding followed by RBD binding are shown in blue, while the reverse order of binding is shown in purple.
