Antibody variants
An antibody and affinity technology, applied in the direction of antibodies, anti-inflammatory agents, anti-animal/human immunoglobulin, etc., can solve the problem of prolonging the residence time of serum
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Embodiment 1
[0213] Example 1 Affinity to TNFα
[0214] method:
[0215] Affinity to TNFα was measured by Biacore. CM5 chips were prepared using standard amine immobilization Biacore procedures. After insertion of the CM5 chip, the system was primed and then normalized using BIA normalization solution (Biacore Preventative Maintenance Kit 2). The chip was added to the system using PBS-T running buffer; the chip surface was perfused with three injections of 50 mM NaOH before fixation. Protein A is immobilized on the chip surface. For this, the protein was diluted to 5 μg / mL in 10 mM acetate buffer, pH 4.5, and then injected to generate a binding reaction of approximately 1000 RU in all 4 flow cells. To remove non-covalently bound material from all chip flow cells, three 15-second 50 mM NaOH washes were performed. On the protein A chip, antibodies were captured in flow cells 2 and 4, and flow cells 1 and 3 were used for reference subtraction. Dilute the test antibody to 10nM in PBS-T...
Embodiment 3
[0226] Example 3 Affinity with Fcγ receptors (CD64, CD32a, CD32b, CD16a, CD16b)
[0227] method:
[0228]Affinity to FcγRs was measured by Biacore. CM5 chips were prepared using standard amine immobilization Biacore procedures. After insertion of the CM5 chip, the system was primed and then normalized using BIA standardization solution (Biacore Preventative Maintenance Kit 2). Chips were added to the system along with phosphate-buffered saline Tween-20 (PBS-T) running buffer; prior to fixation, the chip surface was coated with three injections of 50 mM NaOH. FcγRs were immobilized on the chip surface using a His-tag capture system. Anti-His tag chips were prepared according to the Biacore kit instructions, and about 12000 RU of antibodies were deposited in all 4 flow cells. To remove non-covalently bound material from all chip flow cells, three 30 second washes with 10 mM glycine, pH 1.5, were performed. Fcγ receptors were diluted in PBS-T to a range of 0.5-2 μg / mL and ...
Embodiment 4
[0235] Example 4 Affinity with FcRn
[0236] method:
[0237] SPR was performed using a Biacore 3000 instrument with a CM5 sensor chip coupled to an anti-TNFα IgGl antibody (approximately 500 resonance units (RU)) using amine coupling chemistry as described by the manufacturer. Coupling was performed by injecting 2.0 ug / mL of each protein into 10 mM sodium acetate (pH 4.5) using an amine coupling kit (GE Healthcare). HBS-P buffer (10 mM HEPES, 150 mM NaCl, 0.005% surfactant P20) at pH 7.4 or phosphate buffer (67 nM phosphate buffer, 150 mM NaCl, 0.005% Tween 20) at pH 6.0 was used for running and Dilution buffer. Binding kinetics were determined by injecting titrated amounts (1000-31.2 nM) of His-tagged monomeric human FcRn (hFcRn) on immobilized antibodies at pH 7.4 or pH 6.0. All SPR experiments were performed at 25°C with a flow rate of 40ul / min. Binding data were zeroed and reference cell values were subtracted. The Langmuir 1:1 ligand binding model provided by BI...
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