Surface plasmon resonance (SPR) binding analysis

Binding kinetics and affinities of C1q-recruiting bsAbs were assessed using surface plasmon resonance technology on a Biacore T200 or Biacore 8K instrument (GE Healthcare). A series of analyte concentrations were prepared in HBS-EP running buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA and 0.05% surfactant Polysorbate 20) and injected at a flow rate of 30–50 μl/min for 2–2.5 min over flow cells (FCs) of Series S CM5 sensor chips immobilized with ligand molecules at various densities depending on assay formats. A capture sensor surface was prepared by covalently immobilizing with anti-human IgG F(ab’)2 fragment specific antibody (Jackson Immuno Research), NeutrAvidin (Thermo Fisher) or an anti-his monoclonal antibody (His capture kit, GE Healthcare,) to the chip surface using (1¬Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)/N-hydroxysuccinimide (EDC/NHS) coupling chemistry. Following surface activation, anti-human IgG F(ab’)2, NeutrAvidin or anti-his antibody in coupling buffer (0.1 M acetate buffer, pH 4.5) was injected over the activated chip surface until a resonance unit (RU) signal of about ~10000 RU (anti-human IgG F(ab’)2 polyclonal antibody), ~2000 RU (NeutrAvidin) or ~10000 RU (anti-his monoclonal antibody) was reached. The activated coupled chip surfaces were then washed and treated with 10 mM glycine-HCl, pH 1.5, to remove uncoupled residual proteins. In each format, all experiments were carried out at 37 °C.

In the first format (Supplementary Fig. ^2a), antibodies were diluted into the running buffer and captured on the coupled anti-human IgG F(ab’)2 polyclonal antibody chip surface at ∼250 resonance units (RU) density, while the antigens were the analytes. Following the capture step, a range of concentration of test antigen (20.0 nM to 0.625 nM for C1q protein, and 90 nM to 1.11 nM for IsdB.6xHis protein) were individually injected over C1q-recruiting bsAb captured surfaces for 2–2.5 min. For all ligands, the association rate constant (ka) was determined from data obtained at multiple test ligand concentrations. The dissociation rate constant (kd), which is independent of test ligand concentration, was determined from the change in antigen-bound test ligand RU over time (~5–10 minutes) for C1q and IsdB.6xHis protein, respectively.

In the second format (Supplementary Fig. ^2b), the biotin-C1q protein was captured on the coupled NeutrAvidin where the final ligand density was ~10 RU. Following the capture step, a range of concentrations of bsAb (500 nM to 7.8 nM), were individually injected over biotinylated C1q capture surfaces for 2 min. For all ligands, the ka was determined from data obtained at multiple test ligand concentrations. The kd was determined from the change in antigen-bound test ligand RU over time (~5 minutes) for C1q-recruiting bsAb respectively.

In the third format (Supplementary Fig. ^2c), the IsdB.6xHis protein was captured on the coupled anti-his antibody chip where the final ligand density was ~350 RU. Following the capture step, a range of concentrations of bsAb (50 nM to 12.5 nM), were individually injected over IsdB.6xHis capture surfaces for 2.5 min. For all ligands, the ka was determined from data obtained at multiple test ligand concentrations. The kd was determined from the change in antigen-bound test ligand RU over time (~5 minutes) for C1q-recruiting bsAb, respectively. In addition, binding kinetics was performed for C1q protein (20 nM to 0.625 nM) flowing over complex IsdB.6xHis plus C1q-recruiting bsAb captured surface.

Specific Biacore kinetic sensorgrams were obtained by a double referencing procedure as described by Myszka et al.⁴⁸. Because of the hexameric nature of C1q protein and the presence of multiple potential binding sites in the first format, definitive monovalent binding affinities for C1q protein and bsAb can be challenging to obtain. Very low immobilization densities were used to encourage monovalent binding, and the presence of such interactions were evaluated using a 1:1 Langmuir binding model. The data were then processed, and kinetic analyses performed using Scrubber software (version 2.0, BioLogic Software). The kd and ka were obtained via kinetic fitting, and the equilibrium dissociation constant (K_D) was derived by taking the ratio of kd over ka calculated using the simplest 1:1 binding model.