Luminex Assay for COVID-19 Antibody Detection

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.