4m urea

For avidity ELISA, serum samples were applied to wells in duplicate. After one-hour incubation, one set of samples was incubated with wash buffer and another with 4M urea (Sigma-Aldrich, St. Louis, MO, USA) for 5 min and washed twice with wash buffer, and the ELISA protocol was completed as described. The avidity index (AI) was calculated as previously described32 (link) for the lowest dilution (1:2500) as AI = (U+/U-) ×100, where ‘U+’ is the OD₄₉₀ for wells washed with urea and ‘U-‘ is the OD₄₉₀ for wells washed with PBST.

Recombinant MIF protein was purified from Escherichia coli, as previously described [23 (link),24 (link)]. Briefly, BL21 E. coli strains (RBC Bioscience, Taipei City, Taiwan) were transformed with pET28a-MIF in order to express and purify the fusion protein. Bacterial cultures were induced with 0.4 mM isopropyl β-d-thiogalactoside (IPTG, Duchefa Biochemie, Haarlem, Netherlands). Next, bacterial cultures were sonicated for 10 min. at 4 °C, and then centrifuged at 1600× g for 20 min. at 4 °C. Pellets containing His-tagged MIF (His-MIF) were resuspended in binding buffer (0.5 M NaCl, 20 mM Tris-HCl, 5 mM imidazole) containing 4 M urea (Sigma Aldrich, St. Louis, MO, USA). His-MIF was purified while using Ni-NTA His•Bind Resin (Merck, Darmstadt, Germany) and subsequently eluted using elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, and 250 mM imidazole) containing 4 M urea. Finally, MIF protein was dialyzed in order to remove imidazole, residual salts, and urea.

mAb-A was a purified monoclonal antibody Drug Substance produced in house and formulated at pH 6.0. Trypsin enzymes were obtained from Promega (Madison, WI, USA Cat# V5113). Lys-C was purchased from Fujifilm Wako (Osaka, Japan. Cat# SKR0971). Urea (OmniPur^®, Product# 29700) and Trizma^® hydrochloride solution (Cat# T7943) were obtained from Millipore Sigma (Billerica, MA, USA). LC–MS grade water (Cat# W6-1), acetonitrile (Cat# A955-500), formic acid (Cat# A117-50) and trifluoroacetic acid (Cat# A116-1AMP) were obtained from Fisher Chemical (Fair Lawn, NJ, USA). Bovine Serum Albumin (BSA, Cat# A2058-25G), Sodium phosphate dibasic (Cat# S3264-250G), sodium phosphate monobasic monohydrate (Cat# S5011-500G), and sodium chloride (Cat# S9625-1KG) and guanidine hydrochloride solution (Cat# G7294-100ML), and 4M urea (Cat# 51456-500G) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Iodoacetamide (IAA, Cat# 90034) and dithiothreitol (DTT, Cat# 20291) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). pI markers 7.05 (Cat# 046-032) and 9.99 (Cat# 046-034), 1% methyl cellulose (Cat# 101876), and 500 mM arginine (Cat #042-6910) were obtained from ProteinSimple (San Jose, CA, USA). Pharmalyte 3–10 was obtained from Cytiva (Uppsala, Sweden; Cat# 17045601).