Fig. 4

Optimized CHIKV anti-budding assay. The assay uses a novel CHIKV nLuc reporter virus to screen specifically for antibodies that inhibit viral budding. RD cells were first bulk infected with reporter virus, excess virus washed off after 2 h, the monolayer disrupted, and infected cells seeded onto a 96-well plate at 2.5 × 10⁴ cells/well in 100 µL/well of complete growth medium. 100 µL diluted CHIKV-positive human sera or B-cell supernatant was then transferred to assay wells. All wells, including those with positive inhibition control mAb (2 µg/µL C9) and negative control (media only), were supplemented with 20 mM NH₄Cl. Plates were then incubated for 18 h at 37 °C/5% CO₂, following which 50 µL of supernatant was harvested from each well and transferred to white half-well plates (Greiner Bio-One) for luminescence readings. For all assays, spectrophotometer gain was adjusted to negative control wells