Sulfo n hydroxysulfosuccinimide

An explorative Luminex assay was developed to investigate antigen-specific IgG and IgA in serum and saliva. A recombinant prefusion ectodomain trimer of SARS-CoV-2 S protein, the monomeric RBD of the S protein, and N protein were designed, produced, and purified as previously described (42 (link), 43 (link)). The proteins were covalently coupled to Magplex beads (Luminex) using a two-step carbodiimide reaction at a ratio of 75 μg protein to 12.5 million beads for S, at an equimolar concentration for N, and at 3× the equimolar concentration for RBD. Beads were washed with 100 mM monobasic sodium phosphate, pH 6.2, activated with sulfo-N-hydroxysulfosuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Thermo Fisher Scientific), and incubated for 30 min on a rotator at room temperature (RT). Activated beads were washed 3 times with 50 mM MES (morpholineethanesulfonic acid), pH 5.0, proteins were added, and beads were incubated for 3 h on a rotator at RT. The beads were washed with phosphate-buffered saline (PBS) and blocked with PBS containing 2% bovine serum albumin (BSA), 3% fetal calf serum, and 0.02% Tween 20 at pH 7.0 (PBS-blocking) for 30 min on a rotator at RT. Finally, the beads were washed, stored in PBS containing 0.05% sodium azide at 4°C, and used within 6 weeks.

To measure the binding of IgG to the spike proteins of different VOCs, we covalently coupled pre-fusion stabilized spike proteins to Luminex Magplex beads using a two-step carbodiimide reaction as previously described [33 (link)]. In short, Luminex Magplex beads (Luminex) were washed with 100 mM monobasic sodium phosphate pH 6.2 and activated by addition of Sulfo-N-Hydroxysulfosuccinimide (Thermo Fisher Scientific) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (Thermo Fisher Scientific) and incubated for 30 minutes on a rotator at room temperature. After washing the activated beads three times with 50 mM MES pH 5.0, the spike proteins were added in ratio of 75 μg protein to 12.5 million beads and incubated for three hours on a rotator at room temperature. To block the beads for aspecific binding, we incubated the beads for 30 minutes with PBS containing 2% BSA, 3% fetal calf serum and 0.02% Tween-20 at pH 7.0. Finally, the beads were washed and stored at 4°C in PBS containing 0.05% sodium azide.

To measure the binding of IgG to the S-proteins of different VOCs, we covalently coupled pre-fusion stabilized S-proteins to Luminex Magplex beads using a two-step carbodiimide reaction as previously described^{10 (link)}. In short, Luminex Magplex beads (Luminex) were washed with 100 mM monobasic sodium phosphate pH 6.2 and activated by addition of Sulfo-N-Hydroxysulfosuccinimide (Thermo Fisher Scientific) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (Thermo Fisher Scientific) and incubated for 30 min on a rotator at room temperature. After washing the activated beads three times with 50 mM MES pH 5.0, the S-proteins were added in ratio of 75 µg protein to 12.5 million beads and incubated for three hours on a rotator at room temperature. To block the beads for aspecific binding, we incubated the beads for 30 min with PBS containing 2% BSA, 3% fetal calf serum and 0.02% Tween-20 at pH 7.0. Finally, the beads were washed and stored at 4 °C in PBS containing 0.05% sodium azide^{31 ,34} .