Sephadex g 15

Hydroxypropyl-β-cyclodextrin (HP-β-CD) MW 1380 Da (SR: 0.6), dexamethasone (DMS), thiourea, fluorescein diacetate (FDA), Ellman’s reagent (2,2′-dinitro-5,5′-dithiobenzoic acid), l-cysteine hydrochloride monohydrate, tris(hydroxymethyl)aminomethane, Sephadex G-15, Type II porcine gastric mucin, Dulbecco’s Modified Eagle Medium (DMEM), calf bovine serum, a mixture of antibiotics consisting of an aqueous solution of penicillin (10,000 U/mL) and streptomycin (10,000 µg/mL), 10 mM phosphate buffer pH 7.3 without Ca²⁺ and Mg²⁺ (PBS_A), and a trypsin-EDTA buffer solution containing 0.25% trypsin were all purchased from Sigma-Aldrich (Darmstadt, Germany). Fibroblast BALB/3T3 clone A31 cells (CCL-163) were obtained from American Type Culture Collection.

The stock of a cTn I-T-C complex (SRM 2912) and monoclonal antibodies (clones M18, 560, 19C7, and MF4) specific to cTnI were supplied by Hytest (Turku, Finland). Sodium phosphate dibasic, sodium phosphate monobasic monohydrate, sodium chloride, casein (sodium salt form, extract from bovine milk), 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB), Sephadex G-15, and D-(+)–trehalose dihydrate were purchased from Sigma (St. Louis, MO, USA). The NC membrane (HiFlowPlus HFB13504) and polyester membrane were supplied by Millipore (Billerica, MA, USA). EZ-Link NHS-LC-LC-Biotin, succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), dithiotheritol (DTT), goat anti-mouse IgG, and SuperSignal West Femto Chemiluminescent Substrate for HRP were purchased from Thermo Fisher Scientific (Rockford, IL, USA). Streptavidin and HRP were supplied by Calbiochem (San Diego, CA, USA). The cellulose membrane (17CHR, chromatography grade) and the glass fiber membrane for the supply of conjugate were obtained from Whatman (Maidstone, England) and MDI (Ambala Cantt, India) respectively. Hydrogen peroxide, insoluble TMB, and streptavidin-PolyHRP20 (SA-Poly-HRP) conjugate were obtained from Junsei (Tokyo, Japan), Moss (Pasadena, MD, USA), and Fitzgerald (North Acton, MA, USA) respectively. The grade of all reagents was analytical.

Fa solution (2 mL, 60.0 mg/mL) was separated on a Sephadex G-15 (Sigma-Aldrich, Shanghai, China) column (1 × 100 cm). We used a 0.02 M phosphate (pH 7.0) buffer at the flow rate of 0.15 mL/min for the elution. The eluate was monitored at 280 nm and each fraction was collected and lyophilized.