Amine coupling kit

SPR was carried out to assess the binding affinities of mAb to FcγR using a Biacore T100 system (GE Healthcare). A Series S Sensor CM5 chip (GE healthcare) was primed and normalised with BIA Normalising solution (GE Healthcare). The normalised chip dextran was activated with a 1:1 mixture of EDC (0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) and NHS (0.1 M N-hydroxysuccinimide) (Amine Coupling kit; GE Healthcare) for 10 mins. The mAb ligand was diluted to 25 μg/ml in Acetate pH 5 (GE Healthcare) and ~2000 response units (RU) were immobilised to the CM5 sensor chip flow cells via amine chemistry. Ethanolamine (Amine Coupling kit; GE Healthcare) was used to deactivate excess dextran groups on the chip flow cells. Recombinantly produced FcγR (I, IIA, IIB, IIIA, IIIB) (R&D Systems) were prepared in HBS-EP (GE Healthcare) at 0.16–100 nM (FcγRI) or 1.6–1000 nM (FcγRIIa, IIb, IIIa, IIIb). Kinetic analysis was performed according to the following parameters: sample on/off times 300 s at a flow rate of 30 μl/min with 30 s regeneration of 30 μl/min 10 mM Glycine pH 2.0. FcγR flowed through all cells simultaneously. A blank reference cell was used to be subtracted from antibody-containing flow cells. Kinetic analysis and steady-state affinity calculation were performed using Biacore Evaluation software (GE Healthcare).

Seven-week-old female BALB/c mice were immunized with the purified CD7 recombinant protein. Hybridomas were produced by fusion of spleen cells with SP2/0 cells. Hybridomas were screened using an ELISA, immunofluorescence, and flow cytometry. The mFc domain of the mouse anti-CD7 mAbs were replaced with the hFc domain derived from human IgG1 using genetic engineering. The recombinant mAbs were expressed and purified as described above. The purified mAbs were analyzed through reduced- or non-reduced SDS PAGE. The binding affinity of mAbs to CD7 recombinant protein was measured using Biacore ×100 instrument with a CM5 sensor chip (GE Healthcare, Chicago, IL, USA), according to a previously published procedure [18 (link)]. CD7-mFc was immobilized on CM5 using amine coupling (Amine coupling kit, GE Healthcare). The anti-CD7 mAbs were injected across the chip in a 2-fold dilution series. The equilibrium dissociation constant was obtained by using BIA evaluation 2.0 software. Next, the anti-CD7 mAbs were used as primary antibody to incubate CCRF-CEM cells for 0 h, 1 h, 2 h, 4 h and 6 h, respectively. The CCRF-CEM cells were then stained with FITC-conjugated second antibody, and the flow cytometric assay was performed to assess the levels of internalization.

Surface plasmon resonance measurements using a BIAcore T100 (GE Healthcare Life Sciences) were performed essentially as described by Karlsson et al.^{50 (link)}. In brief, antibodies were coupled to CM3 sensorchips using an amine coupling kit (GE Healthcare Life Sciences) at a surface density of approximately 500 RU and a serial dilution (between 6.25 nM and 25 pM) of glycosylated human PAI-1 (and rat PAI-1 (both Molecular Innovations) in HBS-EP + (GE Healthcare Life Sciences) was passed over the sensorchip surface. The resulting sensorgrams were evaluated using the BIAcore T100 Evaluation software (version 2.0.3, GE Healthcare Life Sciences) to provide kinetic data.

The IgG1 mAb (mAb1) and IgG4 mAb (mAb4) were produced in Bristol-Myers Squibb Company. They were expressed in Chinese hamster ovary (CHO) cells and purified by standard chromatographic steps. Both mAbs were frozen and stored at −80°C in formulation buffer. LC-MS grade water was purchased from Honeywell (Plainview, NY). LC-MS grade acetonitrile was purchased from J.T. Baker (Center Valley, PA). 8 M Guanidine-HCl, premium grade TCEP-HCl, and LC-MS grade formic acid were purchased from Thermo Scientific Pierce (Grand Island, NY). Sequencing grade trypsin was purchased from Promega (Madison, WI). Human Fab capture kit, CM5 Sensor Chip, HBS-EP+ running buffer and amine coupling kit was purchased from GE Healthcare Life Sciences (Piscataway, NJ). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.

The binding kinetics of SCP-2, MFE-2, ADRM1, HR23B and FAK with SQAP and SQP were analyzed on a Biacore 3000 instrument (GE Healthcare, Little Chalfont, UK). SCP-2 (400–547), MFE-2 (598–736) and FAK (910–1052) recombinant peptides were obtained as described in the previous section. The buffer of purified peptides was exchanged to HBS-P (150 mM NaCl, 0.005% Surfactant P20, 10 mM HEPES-NaOH pH 7.4) using a PD 10 column (GE Healthcare, Little Chalfont, UK) and the concentrations were quantified using DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). ADRM1 and HR23B human full length proteins were purchased. All proteins were immobilized onto the surface of a CM5 sensor chip using an amine coupling kit (GE Healthcare, Little Chalfont, UK). GST protein was immobilized on the control flow cell and the generated response units (RU) were subtracted from those of HR23B. Different concentrations of SQAP and SQP diluted in HBS-P (150 mM NaCl, 0.005% Surfactant P20, 10 mM HEPES-NaOH pH 7.4, 5% DMSO) were injected for 120 s at a flow rate of 20 μL/min at 25 °C. Kinetic parameters were determined by analyzing the data using BIAevaluation 4.1 software (GE Healthcare, Little Chalfont, UK).

Surface plasmon resonance experiments were carried out with a Biacore T100 protein interaction analysis system (GE Healthcare, Little Chalfont, UK). RANKL (R&D Systems, 462-TEC-010/CF) protein was covalently coupled to a CM5 sensor chip (GE Healthcare) using an amine coupling kit (GE Healthcare). RANK-N was diluted with PBS-EP + running buffer (3 mM EDTA, 150 mM NaCl, 10 mM HEPES and 0.05% (v/v) Surfactant P20) and then injected over the sensor chip at 30 μl min⁻¹ at 25 °C (contact time: 210 s, dissociation time: 400 s). After each measurement of a binding response (in resonance units, RU), the sensor chip was regenerated using 10 mM glycine–HCl buffer (pH 2.5). The values of dissociation rate constant (k_d), the association rate constant (k_a) and equilibrium dissociation constant (K_D) were derived by simulating the binding curves with a monovalent interaction model using BIA evaluation software (GE Healthcare).