LIPS Assay for Antibody Detection

LIPS assay (Figure S1) was performed according to a protocol previously described by Burbelo et al. [19 (link)] with modification. Renilla antigen was prepared by transfecting 293FT cells with Renilla luciferase-orf3b expression plasmid, followed by lysis at 48 h post-transfection. Heat-inactivated patient sera were 1:100 diluted and incubated with 1×10⁷ light units of Renilla antigen on a rotary shaker. 1.5 µL of Protein A/G resin (Thermo Fisher Scientific, USA) diluted in PBS was then added to each sample and incubated to capture the antibody–antigen complex. Following three washes, the beads were transferred to an opaque 96-well plate. Renilla substrate (Promega, USA) was then added and luciferase signal was measured using the Wallac Victor3 Multilabel Counter (PerkinElmer, USA).