Biaevaluation 4

The binding affinity of recombinant DIII with ZIKV-116 was monitored by BLI using an Octet-Red96 device (Pall ForteBio). ZIKV-116 and ZIKV-116-V.J.Rev mAbs were biotinylated using EZ-Link-NHS-PEG4-Biotin reagent and then loaded onto streptavidin biosensors (ForteBio). Binding at neutral pH was determined in HBS-EP buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20, and 3% BSA). Low pH binding was performed in a similar assay buffer with the pH adjusted to 5.5 using 2-ethanesulfonic acid buffer. Buffer alone with loaded mAb and sensor alone with the highest concentration of DIII were added in parallel to serve as negative controls. The experimental curves were analyzed using Biaevaluation 4.1 (GE Healthcare) and fitted by a 1:1 Langmuir fitting model to determine the associate constant (k_a), dissociate constant (k_d), kinetic binding affinity (K_D, kinetic), and steady-state affinity (K_D, equilibrium).

The binding of FHR-4 to immobilized C3b was measured by surface plasmon resonance using a Biacore 3000 (GE Healthcare). The sensor surfaces were prepared by immobilizing human C3b onto the flow cells of a Biacore series S carboxymethylated dextran (CM5) sensor chip (GE Healthcare) using standard amine coupling and included blank flow cells where no C3b protein was present. Experiments were performed at 25 °C and a flow rate of 15 μl min⁻¹ in PBS with 0.05% surfactant P20. FHR-4 was injected in triplicate at concentrations ranging from 1 to 100 μg ml⁻¹. Samples were injected for 150 s and dissociated for another 200 s; the chip was regenerated with 1 M NaCl for 1 min and re-equilibrated into PBS with 0.05% surfactant P20 prior to the next injection. After subtraction of the blank cell value from each response value, association and dissociation rate constants were determined by global data analysis. All curves were fitted using a 1:1 Langmuir association/dissociation model (BIAevaluation 4.1; GE Healthcare).

Binding affinities of mAbs or BiS3Ab to rhIL-6 were determined with a BIAcore 3000 instrument (GE Healthcare Life Sciences). 100 nm mAb1, mAb2, or BiS3Ab was immobilized on a CM5 chip, and 0.12 nm, 0.37 nm, 1.1 nm, or 3.3 nm rhIL-6 was applied to the chip. Concurrent binding of mAbs and BiSAb to rhIL-6 was demonstrated by BIAcore 3000 instrument (GE Healthcare Life Sciences). 100 nm mAb1 or mAb2 was immobilized on a CM5 chip, and 100 nm rhIL-6 was applied to the chip. Upon stabilization, the same antibody, the second antibody, or BiS3Ab (100 nm) was injected to determine concurrent binding. Experiments were performed at 25 °C in 50 mm sodium phosphate buffer (pH 6), 150 mm NaCl, and 0.05% Tween 20. Data were analyzed using BIAevaluation 4.1 (GE Healthcare Life Sciences), and GraphPad Prism was used to plot the data.

Triggering of SOSIP gp140 Envs by soluble CD4 (sCD4) was monitored via surface Plasmon resonance and was performed on a BIAcore 3000 instrument (GE Healthcare). Antibodies were immobilized using direct immobilization using amine coupling on CM3 sensor chips (GE Healthcare) at ~5000 RU. The SOSIP concentration in the presence and absence of a 1:3 mixture of sCD4 was 200 nM. Samples were injected over at a rate of 30 μl/min for a total of 90 μl using the high performance kinject injection mode with a dissociation phase of 600 s over four flow cells containing 17B, 19B, VRC01, or the Ab82 control. The chip surface was regenerated between measurements using two injections of 20 μl of glycine pH 2.0 at a flow rate of 50 μl/min. The resulting response curves were processed using the BIAevaluation 4.1 (GE Healthcare) software using a double reference subtraction. Reported response values were determined taking the average response from 170 to 175 s after the start of the injection.

The BIAcore 3000 system (GE Healthcare Co. Ltd.) was used to determine the direct protein interaction between the full‐length PTX3 (#1826‐TS; R&D Systems) and the N‐terminal of CD44 (#CD44‐3961H; Creative Biomart). Full‐length PTX3 was immobilised onto a CM5 sensor chip (GE Healthcare Co. Ltd.); the immobilisation level was 4000–6000 response units using amine‐coupling chemistry. Briefly, a mixture of 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide activated the surface of the chip, and then diluted full‐length PTX3 (10 μg/ml in acetate buffer pH 3) was injected into the flow cells for immobilisation. Finally, the remaining activated groups were blocked by ethanolamine. The binding parameters of the experiment were measured over a 1‐min association phase and then followed by a 2‐min dissociation phase at a fluid flow rate of 10 μl/min. Phosphate‐buffered saline (PBS, pH 7.2) was used as the analyte running buffer; 0.05% sodium dodecyl sulphate was used to regenerate the chip surface before the next analyte concentration was applied. The 1:1 Langmuir binding model (BIAevaluation 4.1; GE Healthcare Co. Ltd.) were performed to calculate its corresponding association and dissociation rate constants to determine the affinity.

All interaction experiments were conducted at 25°C in PBST (20 mmol/L phosphate buffer, 0.05% tween 20, 500 mmol/L NaCl, pH 7.4) using an Octet Red 96 instrument (Fortebio). The final volume for all solutions was 200 μL. Biolayer interferometry binding analyses were performed according to standard procedures. Firstly, different concentrations of promiximab-DUBA or promiximab were loaded onto biosensor (Protein A, ForteBio) for 2 min. Secondly, recombinant CD56 ECD was loaded to association with the antibody or ADC for 3 min. Finally, dissociation between promiximab-DUBA or promiximab and CD56 antigen were measured for 16 min. The real-time data were analyzed using Biaevaluation 4.1 (GE Healthcare). Association and dissociation profiles, as well as steady-state equilibrium concentration curves, were fitted using a 1:2 binding model [46 (link)–48 (link)].