N ethyl n 3 dimethylaminopropyl carbodiimide hydrochloride edac

Covalent coupling of oligomeric rPmpD or bovine serum albumin (BSA) (Sigma Aldrich) to carboxylate-modified L4530 fluorescent beads (Sigma Aldrich) was performed by reaction with 1mM N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) (Sigma Aldrich) in 50mM MES pH 6.1. 50 μg/mL, 25 μg/mL, 2.5 μg/mL and 0.25 μg/mL concentrations of rPmpD (or 200 μg/mL BSA) were used for coating beads. The mixture was incubated at 20°C with gentle shaking for 1h and the reaction was stopped by centrifugation of the suspension, discarding the supernatant, and washing the pellet twice in binding buffer (RPMI containing 10% BSA (w/v), 10mM HEPES, pH 7.5) to remove all traces of EDAC. Coupling of rPmpD protein to the beads was verified on SDS-PAGE by inspection of the reaction supernatant and boiling of coated beads at 95°C for 5mins.

Poly(acrylic acid) (PAA) (M_w = 100,000 Da, 35 wt % in H₂O; data provided by supplier) was obtained from Sigma-Aldrich (Poznań, Poland). Coupling agents (N-Ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC; 98%) and 4-N,N’-dimethylaminopyridine (DMAP; 98%)) were purchased from Sigma-Aldrich (Poznań, Poland). Fatty alcohols (reagent grade, 98%)—dodecanol and hexadecanol—were obtained from Reachim (Moscow, Russia) and Fluka (St. Gallen, Switzerland), respectively. All other reagents and solvents were purchased from Avantor Performance Materials (Gliwice, Poland). All materials were used without further purification. All experiments and synthetic/purification procedures were performed using deionized water (Millipore).

Yellow-green fluorescent latex beads (1 µM diameter) (Sigma-Aldrich) were washed twice with a buffer consisting of 50 mM 2-(N-Morpholino)ethanesulfonic-acid (MES) (Sigma-Aldrich) and 1.3 mM N-Ethyl-N′-(3-dimethylaminopropyl)-carbodiimide-hydrochloride (EDAC) (Sigma-Aldrich) pH 6.1. Covalent coating of latex beads was performed by adding reSARS-CoV-2 full-length spike protein His-tag (Acro Biosystems) to beads in MES/EDAC-Buffer. The mixture was incubated for 2 h at 37°C. Thereafter 2% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS was added for blocking.