Tracedrawer

The Sensor Chip NTA (SEN-AU-100-10-NTA, SNC1006, Nicoya) should be prepared before the experiment, which was performed with an OpenSPRTM surface plasmon resonance instrument (Nicoya). The Casp3 (10050-H08E, SinoBiological) and PARP1 (11040-H08B, SinoBiological) proteins were diluted to 30 μg/mL in immobilization buffer (1× PBS, pH = 7.4). Imidazole and NiCl₂ solutions were injected into the activated chip to complete the surface functionalization of the chip. Then, Casp3 and PARP1 proteins were injected at a flow rate of 20 μL/min, and the capture levels were respectively reached at 1000 RU and 1200 RU. Micromolecules were diluted with Running Buffer (1 × PBS with 1% DMSO, pH = 7.4) and injected into the flow cell of the channel at a flow rate of 20 μL/min for an association of 240 s, followed by 300 s dissociation. Both the association and dissociation processes were handled with the Running Buffer. Repeat 5 cycles of analyte according to analyte concentrations in ascending order. After each cycle of interaction analysis, the sensor chip surface should be regenerated completely with PBS as the injection buffer at a flow rate of 100 μL/min for 30 s to remove the ligand and any bound analyte. The analysis software used in this experiment was TraceDrawer (Ridgeview Instruments ab Sweden) and was analyzed by the one-to-one analysis model.

All SPR measurements were performed on a four-channel SPR sensor platform (PLASMON IV) developed at the Institute of Photonics and Electronics (IPE) of the Academy of Sciences of the Czech Republic, Prague. Gold SPR chips were functionalized following the Neburkova et al. protocol (Neburkova et al. 2018 (link)). Afterwards, 500 µl of 0.3 µM biotinylated target 58rRNA in buffer R + 0.1% Triton X-100 solution was loaded on the functionalized chip. Assay with immobilized neutravidin without 58rRNA target served as negative control to all kinetic experiments. CL11 (525 to 66 nM), CL11-M (200 to 21.25 nM), and CL11-E (11 to 2.7 µM) in buffer R + 0.1% Triton X-100 were injected (association phase) for several minutes, and then buffer R + 0.1% Triton X-100 alone was injected (dissociation phase). Obtained data were fitted by the logistic equation using TraceDrawer (Ridgeview Instruments AB), and k_on, k_off, and K_D values were calculated from Kinetic evaluation using OneToOne fitting model.

The binding kinetics against CLDN3 on the cell surface were measured using LigandTracer Green (Ridgeview Instruments AB, Vänge, Sweden). The hCLDN3/HEK293, hCLDN3/TOV-112D, and mCLDN3/HEK293 cells, as positive cells, and HEK293 and TOV-112D cells, as negative cells, were seeded on a limited area of 100 mm culture dish at a density of 3 × 10⁵ cells/mL in 500 μL culture medium, and after 6 h, 10 mL growth medium was added to culture dish. Cells were incubated overnight, and 3 mL of the medium was changed before the experiment. The h4G3 was labeled with DyLight dye 488 using DyLight Antibody Labeling Kits (Thermo Fisher Scientific) following the manufacturer’s instructions. The cell culture dish was clamped onto the device and the fluorescence baseline was recorded. Each time the respective fluorescence reached equilibrium, Dylight dye 488-labeled h4G3 was added stepwise to a final concentration of 3 nM and 9 nM for hCLDN3 cell lines, and 30 nM and 90 nM for mCLDN3 cell lines. In the dissociation phase, the remaining medium was removed, and 3 mL fresh medium was added to the culture dish. All measurements were performed using a 15 s detection time and 4 s detection delay. Recorded data were analyzed by TraceDrawer (Ridgeview Instruments AB).