Proteostat protein aggregation assay

Proteostat Protein Aggregation Assay was purchased from Enzo Life Sciences (ENZ-51035, Farmingdale, NY, USA). Plasma proteins were incubated with the commercial reagent sensitive to protein aggregation at a ratio of 2 μL reagent to 98 μL of diluted plasma samples. Positive and negative controls were included, as indicated. Proteostat Protein Aggregation standards (ENZ-51039, Enzo Life Sciences, Farmingdale, NY, USA), containing stressed and unstressed IgGs, were used to establish a standard curve and calculate the percentage of aggregated protein in test samples. Fluorescence was measured using a Tecan Infinite M200 microplate reader (TECAN, Männedorf, Switzerland), with excitation set at 550 nm and an emission filter of 600 nm. For Thioflavin T (ThT) (211760050, Thermo Fischer Scientific, Waltham, MA, USA) staining, 5 µL of human plasma was loaded onto wells of a 96-well microplate, in duplicate. Samples were then mixed with 10 µM ThT in 100 mM sodium phosphate buffer, pH 7.4, to a final volume of 250 µL per well [29 (link)]. Fluorescence readings were acquired using the Tecan Infinite^® M200 PRO microplate reader at an excitation of 444 nm and emission of 485 nm.

The WT and mutated IL-18 proteins were subjected to buffer exchange using a Vivaspin ultrafiltration unit (Sartorius, Göttingen, Germany) against PBS. To generate IL-18 aggregates, 40 µg/ml of each protein was incubated at 37 °C, 220 rpm for 16–18 h in a shaker incubator. The protein aggregation was monitored by ProteoStat Protein Aggregation Assay (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s protocol. Briefly, the proteins were loaded in triplicate in 96 well fluorescent microplates, followed by the addition of ProteoStat detection dye. The reaction mixture was then incubated for 15 min in the dark at room temperature. The fluorescence signal was determined by excitation at 485 nm and emission at 620 nm using a Varioskan™ LUX multimode microplate reader (Thermo Scientific, USA).

Analysis of aggregated proteins was performed with the PROTEOSTAT® protein aggregation assay (Enzo) according to the manufacturer’s instructions.

Intravenous human IgG solution (10%, 100 mg/mL) was acquired from GC Pharma (formerly Green Cross Corporation, Yongin, Korea). In the subsequent nebulization experiments, IgG was diluted with saline to 1, 10, 20, and 40 mg/mL. A DC protein assay kit and 4–15% Mini-PROTEAN® TGX™ precast Protein Gels were purchased from Bio-Rad Laboratories (Hercules, CA, USA). PROTEOSTAT® Protein aggregation assay and IgG Human ELISA Kit were purchased from Enzo Life Sciences (Farmingdale, NY, USA) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. Five nebulizers, with three different operating types, were used in this study (Table I). The PARI BOY SX + LC SPRINT was used with red and blue nozzles. PZT the same used in VMN-SM1 and VMN-SM3 was provided by KTMED (Seoul, Korea).Table I

Nebulizers Used in the Study

Mode of nebulizer	Model/manufacture	Abbreviation in the study
Jet	PARI BOY SX + LC SPRINT with red nozzle/PARI GmbH, Starnberg, Germany	JN-PARIr
	PARI BOY SX + LC SPRINT with blue nozzle/PARI GmbH, Starnberg, Germany	JN-PARIb
Vibrating mesh	NE-SM1 NEPLUS/KTMED Co., Seoul, Korea	VMN-SM1
	NE-SM3/KTMED Co., Seoul, Korea	VMN-SM3
Static mesh	NE-U150/Omron Healthcare, Kyoto, Japan	SMN-U150