Covalent Coupling of Antigens to Polystyrene Beads

Antigens were covalently coupled to polystyrene beads (SeroMap Beads; Luminex Corporation, Austin, TX) by modifying carboxyl groups on the beads to ester groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (EMD Millipore Calbiochem, USA) in the presence of N-hydroxysulfosuccinimide (sulfo-NHS) (Thermo Scientific Pierce, USA)^{40 (link)}. Ester groups on the beads bind to primary amine groups on antigens to create stable amide covalent bonds. Beads were briefly sonicated in a water bath and washed with 0.1 M sodium phosphate buffer, pH 6.2 (NaP) in preparation for bead activation. Beads were protected from light and rotated for 20 min in NaP with 5 mg/ml each EDC and NHS. After activation, activated beads were washed and suspended in coupling buffer, antigen added, and rotated at room temperature for 2 h. Coupling buffers and antigen amounts were previously determined for each antigen (Supplementary Table 5). After 2 h, antigen-coupled beads were washed with PBS and unreacted sites were blocked with 1% bovine serum albumin (BSA) in PBS for 30 min. Antigen-coupled beads were resuspended in storage buffer (PBS, 1% BSA, 0.05% Tween-20, 0.02% NaN₃, protease inhibitors) and kept at 4 °C until use in assays.