Human antibody capture kit

Human Antibody Capture Kit (Cytiva, BR100839) and CM5 chip (GE Healthcare) were used to make anti-human IgG (Fc) chip for the affinity detection of human IL-2 (Sino Biological, GMP-11848-HNAE) with SD-01. SD-01 in HBS-EP⁺ buffer (Cytiva, BR100669, 33372) was injected over the chip surface for 30 s (speed: 10 μL/min) as ligand. Then the 2-fold dilution gradient (3.9–250 nM) of human IL-2 was injected as analyte with the speed of 30 μL/min. The chip surface was regenerated after each injection by washing with Glycine 1.5 (Cytiva, BR100354) for 30 seconds (speed: 30 μL/min).
His Capture Kit (GE Healthcare, 29234602) and CM5 chip (GE Healthcare) were used to make anti-His tag chip. The his-tagged human IL-2Rα (Sino Biological, 10165-H08H) or IL-2Rβγ (ACROBiosystems, ILG-H5283) was injected over the chip surface and captured as ligand. The 2-fold dilution gradient of tag-free IL-2 (1.56–100 nM, R&D, 202-IL-050/CF), Fc-tagged IL-2 (0.7825–25 nM, ACROBiosystems, IL2-H5269) or IL-2/SD-01 (0.0976–3.125 nM) was injected as analyte with the speed of 30 μL/min. The surface was regenerated after each injection by washing with regeneration buffer (Cytiva, BR100354) for 30 seconds (speed: 30 μL/min) and binding affinities were measured by surface plasmon resonance on a Biacore 8 K (GE Healthcare).

Interaction of the mAbs and BsAbs with the recombinant proteins was monitored using a Biacore T200 instrument (Cytiva). The measurements were performed in PBS supplemented with 0.05% Tween 20 (pH 7.4), at a flow rate of 30 μL/min at 25 °C. The antibodies were captured on a CM5 chip immobilized with anti-human IgG Fc using Human Antibody Capture Kit (Cytiva) following the manufacturer’s instructions. The antibodies (1 μg/mL) were run for 120 s to capture ca. 400 RU. As antigens, the extracellular domains of human TNFR2 and CD30 were subcloned into a pcDNA3-based rabbit IgG Fc-fusion vector, as described previously^{46 (link)}. The Fc-fusion proteins were expressed and purified as the chimeric antibodies described above. The antigen proteins (10 nM) were run for a contact time of 60 s, and dissociation times of 60 s and 180 s for the 1st and 2nd contacts, respectively.

SPR analyses for the recombinant antibodies NIBIC-71, 7G7/λ, and 7G7/κ were carried out using a Biacore T200 instrument (Cytiva) as previously described^{25 (link)}. Briefly, anti–human IgG (Fc) antibodies were immobilized on a Series S Sensor Chip CM5 (Cytiva) using the Human Antibody Capture Kit (Cytiva) and the Amine Coupling Kit (Cytiva). The human mAb 8A7 was used as a negative control in SPR analyses. Each antibody was captured at approximately 150 response units (RUs). SPR sensorgrams were obtained by injecting twofold serial dilutions of SARS-CoV-2 S protein (ECD, His and Flag Tag) (GenScript) ranging from 80 to 0.625 nM in PBS-T. The experimental parameters were as follows: temperature, 25 °C; flow rate, 30 µL/min; contact time, 240 s; and dissociation time, 900 s. The SPR sensorgrams of each antibody were analyzed using Biacore T200 Evaluation Software version 2.0 (Cytiva).

Kinetic analysis of the antibody variants was performed with surface plasmon resonance using Biacore 8K + instrument (Cytiva). A capture assay was used to for the affinity measurement to minimize the avidity effects caused by the bivalent nature of the antibodies. Human antibody capture kit (Cytiva) was used to immobilize Anti-human IgG (Fc) antibody to a CM5 sensor chip (Cytiva) by amine coupling at approximately 6000 RU. The sample antibody was captured at 62.5 ng/mL for 60 s at a flow rate of 10 μL/min, resulting in capture levels between 10 and 25 RU. A 4-fold dilution series of PCSK9 (Acro Biosystems) with concentrations ranging from 50 nM to 12 pM was flowed over the captured IgG surface for 240 s at a flow rate of 50 μL/min with a dissociation time of 1800 s. The surface was regenerated with 3M MgCl₂ between measurement cycles. The association and dissociation values were determined with Biacore Insight Evaluation software using the Langmuir 1:1 global fitting procedure. All samples were run in duplicates.

Binding affinity of Fab against captured mAb was determined by surface plasmon resonance (SPR) on a BIAcore T200 (Cytiva). The running buffer, 10 mM HEPES, 150 mM NaCl, 0.05% v/v Surfactant P20, 3 mM EDTA, pH 7.4 (HBS-EP+, Cytiva, Marlborough, MA, USA) was used for immobilization and reagent dilutions. All binding kinetics were measured at 25 °C.
For each injection cycle, mAbs were first captured in flow cells 2, 3 and 4 with an anti-human Fc antibody (Human Antibody Capture Kit, Cytiva, Marlborough, MA, USA ) immobilized to the sensor chip (Series S CM5, Cytiva, Marlborough, MA, USA ). Flow cell 1 with no captured mAb was used as a reference. Serial dilutions of the Fab, ranging in concentration from 1 to 32 μM, and buffer blanks were injected in multiple cycles over the captured mAbs and reference surfaces for a 60 s association followed by a 180 s dissociation. The surfaces were regenerated with a 30 s injection of 3 M MgCl₂ after each cycle.
Double referenced titration data were globally fit to a 1:1 Langmuir binding model to determine the association rate constant, ka (1/M·s), and the dissociation rate constant, kd (1/s), using the BIAcore T200 Evaluation Software version 2.0 (Cytiva, Marlborough, MA, USA). The equilibrium dissociation constant was calculated as KD (M) = kd/ka.

The binding kinetics of sACE2 (wild-type or mutants) to RBD were analyzed by SPR using a Biacore T200 instrument (Cytiva) in a single-cycle kinetics mode. Anti-human IgG (Fc) antibody was immobilized onto a CM5 sensor chip (Cytiva) using the Human Antibody Capture Kit (Cytiva) according to the method provided by the manufacturer. The RBD-Fc was captured on the measurement cell via the antibody at a density of ~450 RU, while human IgG1-Fc was captured on the reference cell at a density of ~200 RU. The binding was evaluated by injecting various concentrations of sACE2-His solutions in series using PBS containing 0.05% Tween 20 as a running buffer. The runs were conducted at 25 °C employing the following parameters; flow rate of 30 μl/min, contact time of 120 s, and dissociation time of 480 s. After each run, the surface was regenerated by injecting the Regeneration solution contained in the kit for 30 s. The binding curves of the measurement cell were subtracted with those of the reference cell, and used to derive kinetic binding values. The results were evaluated by using Biacore T200 evaluation software version 4.1.