Kge019

To address β2m amyloid fibril formation based on the supersaturation-limited mechanism, we measured the solubility of β2m monomer using an ultracentrifugation method combined with an ELISA assay^{37 (link),57 (link)}. First, β2m solution containing [β2m] = 1.0 mg/mL, [NaPi (pH 7.4)] = 20 mM, [NaCl] = 300 mM, [ThT] = 5 μM was prepared, and then amyloid fibril formation was induced by ultrasonication using the HANABI-2000 instrument. After a sufficient reaction time over 20 h, the solution reached an equilibrium state with amyloid fibrils, which was confirmed by the saturation of ThT fluorescence intensity. The equilibrated solution was ultracentrifugated at 100,000 × g for 1 h, and then, the concentration of β2m monomers in the supernatant was determined by the ELISA method (KGE019, Human beta 2-Microglobulin Parameter Assay Kit, R&D Systems). In the ELISA assay, optical density of the samples was recorded using a microplate reader (SH-9000, Corona Electric Co., ltd.) with SF6 software (Version 5.12.1, Corona Electric Co., ltd.).

After the last administration, the urine of the mice was collected, and the levels of β₂-microglobulin (β₂-MG) (KGE019, R&D Corporation, USA) and retinol binding protein 4 (RBP4) (DRB400, R&D Corporation, USA) were detected.