Mk 150

The levels of the ACR-conjugated protein, FDP-Lys, were measured using a competitive ELISA kit (MK-150, Takara Bio, Shiga, Japan), according to the manufacturer's protocol. The protein levels of sVAP-1/SSAO in the samples were measured using an ELISA kit for human sVAP-1/SSAO (BMS259, Thermo Fisher Scientific, Waltham, MA). The vitreous samples were pre-diluted to 1:10 by assay buffer and the diluted samples (100µL) were used for sVAP-1/SSAO measurement.
For FDP-lys measurement, vitreous samples (50µL) were used without dilution.

Recombinant human VAP-1/SSAO (rhVAP-1/SSAO, Peprotech, Rocky Hill, NJ) was incubated with 2.5mM spermine (S4264, Sigma) in each of the following buffers: 50mM HEPES (pH 7.4), 50mM Tris-HCl (pH 7.4), physiological HEPES (50mM HEPES, 140mM NaCl, 5mM KCl, 2mM CaCl 2 , 1.4mM MgCl 2 , pH 7.4), and PBS (137mM NaCl, 8.1mM Na 2 HPO 4 , 2.7mM KCl, 1.5mM KH 2 PO 4 ; pH 7.4) at 37°C. For the inhibition assay, rhVAP-1/SSAO was incubated with 2.5mM spermine in PBS, with or without 100µM semicarbazide (Cell technology, Fremont, CA) or 100nM of the RTU-1096. The concentration of hydrogen peroxide was measured using a Fluoro hydrogen peroxide kit (Cell technology), according to the manufacturer's protocol. The FDP-Lys concentration was also measured using an ELISA kit (MK-150, Takara Bio), when rhVAP-1/SSAO was incubated with 2.5mM spermine and 1mg/mL human serum albumin in PBS, with or without 100µM semicarbazide or 100nM RTU-1096, at 37°C.

Cells were washed twice with PBS and incubated with 200µM spermine in PBS, with or without 100µM semicarbazide or 100nM RTU-1096, and the concentration of hydrogen peroxide in the supernatant was measured. For FDP-Lys detection, cells were washed twice with PBS and incubated with 200µM spermine and 1mg/mL human serum albumin in PBS at 37°C, and the concentration of FDP-Lys was measured using an ELISA kit (MK-150, Takara Bio).