Ez link sulfo nhs lc biotin kit

Affinities were determined with ForteBio-based biolayer interferometry (Pall: Octet^RED96). Sintilimab was biotinylated with EZ-Link Sulfo-NHS-LC-Biotin kit (Thermo Scientific), and loaded onto SA - Streptavidin biosensors at 150 μg/ml. After washing, sensors were dipped into buffer containing antigen at 5 μg/ml or the indicated concentrations, then dissociated in SD buffer (sample dilution buffer: 1× Phosphate Buffered Saline
(PBS) + 0.1% BSA + 0.05% Tween-20). Data analysis was carried out on ForteBio software. Biotinylated FcγRs were loaded onto SA - Streptavidin biosensors and dipped into IgG in solution. For C1q binding, biotinylated anti-PD-1 antibody was loaded onto SA - Streptavidin biosensors and dipped into C1q.
Surface plasmon resonance (SPR) analysis was carried out using Series sensor S chips (protein A; GE Healthcare Life Sciences) for measuring affinity kinetics between IgG and hPD-1 antigen. HBS-EP buffer (running buffer; 0.01 M HEPES, 0.15 m NaCl, 3 mm EDTA, 0.05% v/v P20, pH 7.4) and regeneration buffer (10 mm glycine-HCl, pH 1.5∼2.0) were used throughout experimentation. Antibodies were diluted in running buffer to 5 μg/ml. Antigens were prepared and serially diluted 2× from 25 nm (human) or 50 nm (cynomolgus) to 0.78 nm. Antibody was immobilized onto the chip with an/tigen flowed across the chip in running buffer.

We coated 96-well Nunc-Immuno Maxi-Sorp plates (Thermo Scientific) with recombinant human IGF1R ECD (Sino biological, 100 ng/well) for 16 h at 4°C. The plates were then washed with PBS-T (Tween 20, 0.05%) and blocked with 1% BSA in PBS (200 μL/well) for 2 h at 37°C. Serially diluted M30103, B30104, or A12 (400 nM–0.026 nM) was added to each well (100 μL/well) and incubated for 30 min at 37°C. PBS was used as a negative control. After washing five times with PBS-T, human IGF1 that was biotinylated using the EZ-Link Sulfo-NHS-LC-Biotin kit (Thermo Fisher Scientific) was added into each well (100 ng/well) and incubated for 2 h at 37°C. The plates were further incubated with streptavidin-horseradish peroxidase (Pierce, Cat: #21126, 1:5,000 from 1 mg/mL stock, 20 ng/well) for 1 h at 37°C. After washing with PBS-T, the plates were incubated with 100 μL of TMB substrate reagent (Sigma) for 5 min. The calorimetric reaction was measured using a microplate reader (Molecular Device SpectraMax 190) at 450–650 nm after the addition of 50 μL of 1 N sulfuric acid.